Methods of use of epsilon inhibitor compounds for the attenuation of pain

ABSTRACT

The disclosure herein relates to modified εPKC inhibitory peptides, methods of generating such peptides, and method for using εPKC inhibitory peptides for the treatment of pain.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/018,111, filed Jan. 22, 2008, now U.S. Pat. No. 8,017,583, nowallowed, which claims the benefit of priority of U.S. ProvisionalApplication Nos. 60/881,396, 60/903,684, 60/917,876, and 60/977,332, allof which are incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Jun. 8, 2011, and named“632008008US04seqlist.txt” (19,938 bytes), the contents of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

This present disclosure relates to the development and use ofcompound(s) that modulate different types of pain, and where compoundsthat may have overlapping and/or non-overlapping mechanisms ofbiochemical activity are incorporated into a single compound entity(so-called “Hybrid” compounds or peptides) for the treatment of pain andrelated conditions. Wherein the compounds comprise one or more epsilonPKC (εPKC) inhibitory peptides coupled to at least one carrier moietyand where the inhibitory peptides, the carrier moiety, or both have beenmodified from a prototype sequence to increase the stability, potency,or both of the resulting compound. The εPKC inhibitory peptides may alsobe coupled to one or more modulatory peptides with specific activityagainst one or more of the other PKC isozymes, including PKC alpha,beta, delta, gamma, theta or eta. The benefits of a hybrid compound orpeptide over an isozyme-specific PKC modulator would be to provide abroader spectrum of activities to modulate various types of pain and/orprovide greater potency and/or greater safety for a pain modulatingcompound and/or to provide dual therapeutic activities to alleviatemultiple aspects of a disease condition (e.g., combining pain-reducingactivity with an anti-inflammatory activity).

BACKGROUND

Pain is an uncomfortable sensation caused by inflammation, nerve injury,or overly-sensitive tissue reacting to mechanical, thermal or chemicalstimuli. It is a major health problem: every year many work days arelost due to pain-related conditions. Among a wide variety of pains,neuropathic pain is a disease arising from nerve damage and affects >1million Americans. This condition arises from a variety of causesincluding diabetes, herpes zoster infection (chicken pox/shingles),traumatic nerve injury, cancer, or treatment of cancer withchemotherapeutic agents. Inflammatory pain is another kind of pain thatconstitutes the single largest category due to its multiple etiologies.The search for new analgesic therapy is an area of great interest to themedical community (Reichling & Levine, 1999) because it is associatedwith other local or systemic diseases, such as auto-immune disorder likerheumatoid arthritis and because it is chronic and thus causes prolongedsuffering.

Most of the current pain treatment use remedies ofsystemically-administered drugs, such as non-steroidal anti-inflammatorydrugs (NSAIDS) or opioids. Many of these drugs cause systemic sideseffects ranging from the increase in heart risk to addiction. There areonly a few pain remedies that use local routes of administration, suchas capsaicin cream, which does not work on all kinds of pain and causeslocal irritation (burning sensation, skin pain, skin inflammation, etc).

Protein kinase C (“PKC”) is a key enzyme in signal transduction involvedin a variety of cellular functions, including cell growth, regulation ofgene expression, and ion channel activity. The PKC family of isozymesincludes at least 11 different protein kinases that can be divided intoat least three subfamilies based on their homology and sensitivity toactivators. The families are the classical, the novel, and the atypicalsubfamilies. Each isozyme includes a number of homologous (“conserved”or “C”) domains interspersed with isozyme-unique (“variable” or “V”)domains. Epsilon PKC is a member of the “novel” subfamily, along with δ,η and θPKC. Members of this subfamily typically lack the C2 homologousdomain and do not require calcium for activation. Individual isozymes ofPKC have been implicated in the mechanisms of various disease states.Epsilon PKC inhibitory peptides derived from εPKC have been generatedand shown to impact nociception. For example, see U.S. Pat. Nos.6,376,467 and 6,686,334.

One problem with this approach is that the “naked” termini of theexcised fragments are different from their context in the protein,revealing free amine and carboxyl groups at the points where thefragment attaches to the remainder of the protein. These extraneousmoieties may render the peptide more susceptible to proteases. As aresult of these liabilities the potency of the peptide may be less thandesired and the in vivo half-life may be significantly shortened.

A second area of the prior art makes use of a similar strategy, wherein“carrier” peptides are designed as fragments of HIV-Tat and otherproteins. These peptide fragments mimic the ability of the parentprotein to cross cell membranes. Of particular interest is the propertythat “cargo” peptides can be attached to these carrier peptides suchthat both cargo and carrier peptides are carried into the cell by thesecarrier peptide fragments.

Recognizing that the carrier peptides are fragments, similardeficiencies may apply as noted above for the cargo peptides. That is,the exposed termini may confer undesirable properties including proteasesusceptibility.

Prior art cargo/carrier peptide constructs have made use of a Cys-Cysdisulfide bond between cargo and carrier, which can be cleaved by anumber of agents, such as glutathione reduction when the peptides entercells. This property has been thought to be important for biologicalactivity, since the physical separation of cargo and carrier allows thetwo moieties to exert their independent effects within the cell.However, this hypothesis has not been convincingly tested, andnon-cleavable analogs may in fact have good activity. Further, thedisulfide bond is cumbersome to assemble, and prone to chemicaldegradation.

The design of certain prior art cargo/carrier peptides is based on acontiguous sequence of amino acids from the protein. However, theoptimal length of the peptide has not yet been well defined, being basedon sequence comparison analysis and theoretical prediction of thedesired sequence rather than on an empirical basis of analog testing.Thus, increased potency may be anticipated from analogs of thepreviously described cargo peptides which contain additional residuescorresponding to the εPKC domain from which the have been derived.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of a modified εPKC inhibitorypeptide (KP-1634) (SEQ ID NOS:49,54,56).

FIG. 2 shows the chemical formula of an inhibitory εPKC inhibitorypeptide (KP-1586).

FIG. 3 shows the impact of rat or human sera on the stability ofpeptides KP-1586, KP-1630, and KP-1631, by plotting the relativeconcentration of test peptides over time.

FIG. 4 shows the impact of rat or human sera on the stability ofpeptides KP-1632, KP-1633, and KP-1634, by plotting the relativeconcentration of test peptides over time.

FIG. 5 shows the impact of rat or human sera on the stability ofpeptides KP-1635, KP-1636, and KP-1637, by plotting the relativeconcentration of test peptides over time.

FIG. 6 shows the impact of time and temperature on the chemicalstability of peptides KP-1586, KP-1630, KP-1631, KP-1632, KP-1633,KP-1634, KP-1635, KP-1636, KP-1637, and KP-1638, by plotting therelative concentration of test peptides over time.

FIG. 7 shows the results of a formalin test to show the attenuation ofacute pain in a plot of flinches per minute versus time in rats treatedwith control and two doses of KP-1586.

FIG. 8 shows the impact of εPKC inhibitory peptide on acute inflammatorypain in a bar graph plotting paw withdrawal latency for peptide KP-1586and control peptide KP-1587 in a carrageenan model.

FIG. 9 shows a bar graph plotting paw withdrawal latency and comparesimpact of inhibitory peptide KP-1586 or control peptide KP-1587 in achronic inflammatory pain model in rats where carrageenan isadministered followed 5 days later by PGE₂ to promote chronichyperalgesia.

FIG. 10 shows a line plot indicating the effect of a εPKC inhibitorypeptide on paw withdrawal threshold measurements.

FIG. 11 shows bar graphs of the impact of subcutaneous infusion of aεPKC inhibitory peptide on thermal hyperalgesia in rats following L5nerve transection (a neuropathic pain model).

FIG. 12 shows a line plot indicating the impact of a subcutaneous bolusof a εPKC inhibitory peptide on thermal hyperalgesia in rats followingL5 nerve transection measured using paw withdrawal latency.

FIG. 13 shows a bar graph indicating the impact of subcutaneous infusionof a εPKC inhibitory peptide on thermal hyperalgesia in rats followingL5 nerve transection.

FIG. 14 Effects of local administered εPKC inhibitors onλ-carrageenan-induced hyperalgesia in response to mechanicalstimulation. Increases in paw-withdrawal threshold (PWT) upon injectionof εV₁₋₂ via intraderminal (A) and subcutaneous (B) routes. An increasein PWT indicates less pain. An increase in PWT back to the levels beforecarrageenan referred to anti-hyperalagesic effect. An increase in PWTabove the pre-carrageenan levels implicated a potential analgesiceffect.

FIG. 15. Effects of unilateral de-afferentation on anti-hyperalgesiceffects of εPKC inhibitors administered to the limb on the same side ofsurgery, distal from the nerve transaction. Intradermal (i.d.)administration of the compound on the ipsilateral paw was still able toinhibit the carageenan-induced pain in the de-afferenated animal.

FIG. 16. Effects of bilateral lumbar sympathectomy plus bilateralsuprarenal ganglionectomy on anti-hyperlagesic effects of εPKCinhibitors. Experiment was conducted 7-days after surgery to allowdegeneration of the SPGN (Sympathetic post-ganglionic neurons)terminals. Surgical sympathectomy mimicked the effects of the unilateralde-afferentation surgery. Subcutaneous injection of an εPKC inhibitor(εV₁₋₂-TAT) no longer induced anti-hyperalgesic/analgesic effects in thesympathectomized rats but its effects on the sham surgery animalremained intact. The reversal of hyperalgesic is very rapid within fiveminutes of administering the εPKC inhibitor.

FIG. 17. Effects of injection of phentolamine on anti-hyperalgesiceffects of εPKC inhibitors. Injection of phentolamine, which is anon-selective α-adrenoceptor antagonist, abolished the pain-reducingeffects of εPKC inhibitor (εV₁₋₂-TAT) injected to the distal aspect ofthe limb injected previously with phentolamine. After phentolamine, theanti-hyperalgesic effects of εV₁₋₂-TAT was lost.

FIG. 18: Effects of Intradermal KAI-1678 on Mechanical Hyperalgesia inthe Carrageenan Inflammatory Pain Model in Rats. Rats were treated withan intradermal bolus injection of KAI-1678 60 minutes after carrageenaninjection into the plantar side of the right hindpaw. KAI-1678 was dosedat 10 mcg/kg (triangles) or 100 mcg/kg (circles) to either theipsilateral hindlimb (i.e. the same limb that received carrageenan,filled symbols) or the contralateral hindlimb (i.e. the limb that didnot receive carrageenan, open symbols). Data are presented as mean±SEM(standard error of the mean) for the (PWT) measurements for the animalsin each group (N=2-5 animals/group) at a given time point. The dottedline at ˜90 g represents the pre-carrageenan baseline PWT measurement;PWT indicative of the disease state is ˜60 g. Measurements at or abovethe pre-carrageenan baseline level indicate complete reversal ofcarrageenan-induced mechanical hyperalgesia.

FIG. 19: Effects of Subcutaneous Bolus Administration of KAI-1678 onMechanical Hyperalgesia in the Carrageenan Inflammatory Pain Model inRats. A subcutaneous bolus injection of KAI-1678 or KP-1723, theinactive analog of KAI-1678, was administered to rats 60 minutes aftercarrageenan injection into the plantar side of the right hindpaw.KAI-1678 or KP-1723 was dosed at the indicated doses to thecontralateral hindlimb (i.e., the limb that did not receive thecarrageenan). Data are presented as mean±SEM (standard error of themean) for the (PWT) measurements for the animals in each group (N=2-5animals/group) at a given time point. The dotted line at ˜90 grepresents the pre-carrageenan baseline PWT measurement; PWT indicativeof the disease state is ˜62 g. Measurements at or above thepre-carrageenan baseline level indicate complete reversal ofcarrageenan-induced mechanical hyperalgesia.

FIG. 20: Effect of Dosing Location on Activity of Subcutaneous BolusAdministration of KAI-1678 in the Rat Carrageenan Inflammatory PainModel. Rats injected with carrageenan into the plantar surface of theright hindpaw were given two 10 mcg/kg subcutaneous bolus injections ofKAI-1678 four hours apart beginning 60 minutes after carrageenaninjection. The two doses of KAI-1678 were made to different sites on therats as indicated. Data are presented as mean±SEM (standard error of themean) for the (PWT) measurements for the animals in each group (N=2animals/group) at a given time point. The dotted line at ˜90 grepresents the pre-carrageenan baseline PWT measurement; PWT indicativeof the disease state is ˜60 g. Measurements at or above thepre-carrageenan baseline level indicate complete reversal ofcarrageenan-induced mechanical hyperalgesia.

FIG. 21: Effects of Subcutaneous Infusion of KAI-1678 on MechanicalHyperalgesia in the Carrageenan Inflammatory Pain Model in Rats. Ratswere treated with a 6-hour subcutaneous infusion of KAI-1678 or KP-1723,the inactive analog of KAI-1678, 60 minutes after carrageenan injectioninto the plantar side of the right hindpaw. KAI-1678 or KP-1723 wasadministered at the indicated dose rates to the contralateral hindlimb(i.e. the limb that did not receive the carrageenan). Data are presentedas mean±SEM (standard error of the mean) for the (PWT) measurements forthe animals in each group (N=2-6 animals/group) at a given time point.The dotted line at ˜90 g represents the pre-carrageenan baseline PWTmeasurement; PWT indicative of the disease state is ˜55 g. Measurementsat or above the pre-carrageenan baseline level indicate completereversal of carrageenan-induced mechanical hyperalgesia.

FIG. 22: Effects of Intravenous Infusion of KAI-1678 on MechanicalHyperalgesia in the Carrageenan Inflammatory Pain Model in Rats. Ratswere treated with an intravenous infusion of KAI-1678 starting 60minutes after carrageenan injection into the plantar side of the righthindpaw. KAI-1678 was infused via the jugular vein at the indicated doserates for either 2 hours (triangles) or 5 hours (circles). After the endof the 5-hour infusion, 10 mg/kg indomethacin was administered by oralgavage to test the responsiveness of the model. Data are presented asmean±SEM (standard error of the mean) for the (PWT) measurements for theanimals in each group (N=3 or 4 animals/group) at a given time point.The dotted line at ˜90 g represents the pre-carrageenan baseline PWTmeasurement; PWT indicative of the disease state is ˜55 g. Measurementsat or above the pre-carrageenan baseline level indicate completereversal of carrageenan-induced mechanical hyperalgesia.

FIG. 23: Effect of Cutting the Sciatic and Saphenous Nerves on theActivity of KAI-1678 in the Carrageenan Inflammatory Pain Model in Rats.A 1-cm section of the sciatic and saphenous nerves was surgicallyremoved from the left (contralateral) hindleg of rats. The next day,carrageenan was injected into the plantar side of the right hindpaw ofthe surgically-treated rats. Sixty minutes after carrageenan injection,a 4-hour subcutaneous infusion of KAI-1678 at 25 mcg/kg/hr was initiatedon the contralateral limb at a site proximal (open circles) or distal(closed circles) to the site of the nerve transection. Data arepresented as mean±SEM (standard error of the mean) for the (PWT)measurements for the animals in each group (N=4 animals/group) at agiven time point. The dotted line at ˜90 g represents thepre-carrageenan baseline PWT measurement; PWT indicative of the diseasestate is ˜60 g. Measurements at or above the pre-carrageenan baselinelevel indicate complete reversal of carrageenan-induced mechanicalhyperalgesia.

FIG. 24: Effect of Surgical Sympathectomy on the Activity ofSubcutaneously Administered KAI-1678 on Mechanical Hyperalgesia in theCarrageenan Inflammatory Pain Model in Rats. One week followingbilateral lumbar sympathectomy and suprarenal ganglionectomy,carrageenan was injected into the plantar side of the right hindpaw ofsurgically treated rats (Sympathectomized animals—closed circles) orrats in which the lumbar sympathetic chain and suprarenal ganglia wereexposed but not removed (Sham surgery animals—open circles). Sixtyminutes after carrageenan injection, a 4-hour subcutaneous infusion ofKAI-1678 at 25 mcg/kg/hr was initiated into the hindlimb of each animal,contralateral to the site of carrageenan administration. Data arepresented as mean±SEM (standard error of the mean) for the (PWT)measurements for the animals in each group (N=2-4 animals/group) at agiven time point. The dotted line at ˜90 g represents thepre-carrageenan baseline PWT measurement; PWT indicative of the diseasestate is ˜55 g. Measurements at or above the pre-carrageenan baselinelevel indicate complete reversal of carrageenan-induced mechanicalhyperalgesia.

FIG. 25: Effects of Subcutaneous Bolus Administration of KAI-1678 onMechanical Allodynia in the L5 Transection Neuropathic Pain Model inRats. Seven days after transection of the L5 spinal nerve, rats weregiven a subcutaneous bolus of KAI-1678 at the indicated doses (timezero). At the indicated times after compound administration, animalswere tested with von Frey filaments to determine (left) the number ofpaw withdrawals out of 30 tests (5 tests at each filament) or (right)the paw withdrawal threshold determined as the lowest von Frey filamentthat produced at least 3 withdrawals out of 5 tests. Data are presentedas mean±SEM (standard error of the mean) for the animals in each group(N=6 animals/group) at a given time point.

FIG. 26: Effects of Subcutaneous Infusion of KAI-1678 on Allodynia inthe L5 Transection Neuropathic Pain Model in Rats. The day aftertransection of the L5 spinal nerve, osmotic minipumps containingKAI-1678 were implanted subcutaneously to deliver the indicated dailydoses of compound. At the indicated times, animals were tested (left)with von Frey filaments to determine the number of paw withdrawals outof 30 tests (5 tests at each filament) or (right) the paw withdrawallatency in response to exposure to a radiant heat source. Data arepresented as mean±SEM (standard error of the mean) for the animals ineach group (N=6 animals/group) at a given time point.

FIG. 27: KAI-1678 Plasma Concentrations in Rats After IV Bolus. Plasmaconcentrations of KAI-1678 after intravenous bolus administration at 300and 3,000 mcg/kg. Averaged data from three rats (300 mcg/kg) and tworats (3,000 mcg/kg) are shown. A preliminary estimation of terminalhalf-life from these data is ˜38 and ˜66 minutes (300 mcg/kg and 3,000mcg/kg, respectively).

FIG. 28: KAI-1678 Plasma Concentrations in Rats Dosed by IV Infusion.Plasma concentrations of KAI-1678 administered as an intravenousinfusion at 50 mcg/kg/hr. Averaged data from three rats are shown.

FIG. 29: KAI-1678 Plasma Concentrations in Rats after Subcutaneous BolusInjection. Plasma concentration of KAI-1678 after subcutaneous bolusadministration at approximately 80 and 800 mcg/kg. Averaged data fromthree rats (80 mcg/kg) and four rats (800 mcg/kg) are shown. Apreliminary estimation of terminal half-life from these data is ˜35minutes.

FIG. 30: KAI Plasma Concentrations in Rats Dosed by SubcutaneousInfusion. Plasma concentrations of KAI-1678 in rats dosed bysubcutaneous infusion for two hours. Averaged data from two rats at eachdose level are shown.

FIG. 31: KAI Plasma Concentrations in Dogs Dosed by SubcutaneousInfusion over 5 Days. Plasma concentrations of KAI-1678 in dogs dosed bysubcutaneous infusion at 3, 8 and 25 mg/kg/day. Note that samples wererecovered on Day 1 for the first four hours and on Day 6 at the end ofinfusion (EOI), with no sampling in the period between.

DISCLOSURE OF THE INVENTION

The disclosure herein relates to modified εPKC inhibitory peptides,methods of generating such peptides, and method for using εPKCinhibitory peptides for the treatment of pain. The disclosed inventionalso relates to the role of locally-administered protein kinase Cepsilon (εPKC) inhibitors play in suppressing pain perception. Methodsto suppress pain systemically with an εPKC inhibitor, particularlythrough mechanisms that require affecting primary afferent function andmodulation of the sympathetic nervous system. Hybrid peptides comprisingan εPKC-specific inhibitor and another PKC modulatory peptide are alsocontemplated as falling within the scope of the presently disclosedinvention. Any PKC modulatory peptide can be used to prepare the hybridconstruct so that the activity of more than one isozyme-specific PKCmodulator is combined into a single hybrid compound/peptide. Otheraspects and embodiments will be apparent to those skilled in the artform the following detailed description.

DESCRIPTION OF THE INVENTION

The presently described invention relates to modified peptides whichinhibit the epsilon protein kinase C (εPKC) isozyme and are coupled toanother isozyme-specific PKC modulator. Typically, the εPKC inhibitorypeptides discussed herein are coupled to a carrier moiety to facilitatetransport of the inhibitory peptide to a target cell. The cargoinhibitory peptide, the carrier peptide, or both can be modifiedrelative to a prototype control to increase the stability of theresulting cargo/carrier peptide constructs. The disclosed modified εPKCpeptides are useful in preventing, reversing and otherwise treatingvarious types of pain, such as acute pain, chronic pain, neuropathicpain and inflammatory pain. The εPKC inhibitory proteins can also beused to construct a hybrid peptide construct, which comprises one ormore isozyme-specific PKC modulatory peptides with activity againstother PKC isozymes.

DEFINITIONS

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

A “PKC modulatory compound” is any compound, including small moleculesand peptides, which is capable of modulating the enzymatic activity of aPKC isozyme. The term “modulation” refers to increasing or decreasingthe enzymatic activity and other functional activities of a PKC isozyme.A specific PKC modulator (“isozyme-specific PKC modulator”) is anycompound which measurably modulates, either positively or negative, onePKC isozyme over another.

A “PKC activator” is any compound, including small molecules andpeptides, which is capable of activating the enzymatic activity of a PKCisozyme. A specific PKC activator is any compound which measurablyactivates one PKC isozyme over another.

A “PKC inhibitor” is any compound, including small molecules andpeptides, which is capable of inhibiting the enzymatic activity andother functional activities of a PKC isozyme. A specific PKC inhibitoris any compound which measurably inhibits one PKC isozyme over another.

An “εPKC activator peptide” refers to a peptide that can activate anεPKC enzyme.

An “εPKC inhibitory peptide” refers to a peptide that can inhibit orinactivate an εPKC enzyme.

An “γPKC activator peptide” refers to a peptide that can activate anγPKC enzyme.

An “γPKC inhibitory peptide” refers to a peptide that can inhibit orinactivate an γPKC enzyme.

The term “KAI-1586” refers to an peptide derived from the first variableregion of εPKC conjugated via a Cys-Cys disulfide linkage to a “capped”HIV Tat-derived transporter peptide, and can be represented as follows:

The term “KAI-1634” refers to two modified εPKC peptides derived fromthe first variable region of εPKC, which are covalently linked and acapped HIV Tat-derived transporter peptide. The construct is depicted inFIG. 1.

The term “capped” refers to a peptide that has been chemically modifiedto alter the amino terminus, carboxy terminus, or both. A capped carrierpeptide disulfide bonded to an unmodified cargo peptide is shown in FIG.2.

The term “carrier” refers to a moiety that facilitates cellular uptake,such as cationic polymers, peptides and antibody sequences, includingpolylysine, polyarginine, Antennapedia-derived peptides, HIV Tat-derivedpeptides and the like, as described, for example, in U.S. Pats. Nos.4,847,240, 5,888,762, 5,747,641, 6,593,292, US2003/0104622,US2003/0199677 and US2003/0206900. An example of a carrier moiety is a“carrier peptide,” which is a peptide which facilitates cellular uptakeof an εPKC inhibitory peptide which is chemically associated or bondedto the transporter peptide.

The term “prophylaxis” is intended as an element of “treatment” toencompass both “preventing” and “suppressing” as defined herein. It willbe understood by those skilled in the art that in human medicine it isnot always possible to distinguish between “preventing” and“suppressing” since the ultimate inductive event or events may beunknown, latent, or the patient is not ascertained until well after theoccurrence of the event or events.

The term “stability” refers generally to modifications that improveshelf-life times, for example, retarding shelf life-based cys-cysexchange, by retarding proteolytic degradation, or both. The term“potency” relates to the amount of a particular peptide compositionrequired to achieve a particular result. One peptide composition is morepotent than another when dosages of the composition can be reduced toachieve a desired end point. Certain modifications of a given peptidecomposition can be made with improve potency of that composition.

Epsilon Protein Kinase C (εPKC) Inhibitors

There are many known inhibitors of εPKC that can be used in the instantinvention. Small molecule inhibitors of PKC are described in U.S. Pat.Nos. 5,141,957, 5,204,370, 5,216,014, 5,270,310, 5,292,737, 5,344,841,5,360,818, 5,432,198, 5,380,746, and 5,489,608, (European Patent0,434,057), all of which are hereby incorporated by reference in theirentirety. These molecules belong to the following classes:N,N′-Bis-(sulfonamido)-2-amino-4-iminonaphthalen-1-ones;N,N′-Bis-(amido)-2-amino-4-iminonaphthalen-1-ones; vicinal-substitutedcarbocyclics; 1,3-dioxane derivatives;1,4-Bis-(amino-hydroxyalkylamino)-anthraquinones;furo-coumarinsulfonamides; Bis-(hydroxyalkylamino)-anthraquinones; andN-aminoalkyl amides,2-[1-(3-Aminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide,2-[1-[2-(1-Methylpyrrolidino)ethyl]-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide,Gö 7874. Other known small molecule inhibitors of PKC are described inthe following publications (Fabre, S., et al. 1993. Bioorg. Med. Chem.1, 193, Toullec, D., et al. 1991. J. Biol. Chem. 266, 15771, Gschwendt,M., et al. 1996. FEBS Lett. 392, 77, Merritt, J. E., et al. 1997. CellSignal 9, 53., Birchall, A. M., et al. 1994. J. Pharmacol. Exp. Ther.268, 922. Wilkinson, S. E., et al. 1993. Biochem. J. 294, 335., Davis,P. D., et al. 1992. J. Med. Chem. 35, 994), and belong to the followingclasses: 2,3-bis(1H-Indo)-3-yl)maleimide (Bisindolylmaleimide IV);2-[1-(3-Dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide(Go 6983);2-{8-[(Dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide(Ro-32-0432);2-[8-(Aminomethyl)-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide(Ro-31-8425); and3-[1-[3-(Amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimideBisindolylmaleimide IX, Methanesulfonate (Ro-31-8220) all of which arealso hereby incorporated by reference in their entirety.

Epsilon Protein Kinase C (εPKC) Inhibitory Peptides

Various εPKC inhibitors are described herein and can be used with thepresently disclosed methods. The inhibitory peptide can be derived fromany domain, whether variable or constant. Thus, inhibitory peptides canbe derived from V1, V2, V3, V4, or V5. Inhibitory peptides can also bederived from the constant regions C1 (C1a, C1b), C3, C4, or C5. Peptidesoverlapping one or more of these regions are also contemplated. Anothersource of prototype peptides can be found in U.S. patent applicationSer. No. 11/011,557, entitled, “Isozyme-specific antagonists of proteinkinase C,” which is hereby incorporated by reference in its entirety.

In one embodiment, the cargo peptide is an εPKC inhibitory peptidederivative of εV1-2 comprising the amino acid sequence ofE-A-V-S-L-K-P-T (SEQ ID NO:58), a cysteine residue located at the aminoor carboxy terminal ends of the peptide, or internally, and a carrierpeptide linked to the cargo peptide. The cargo peptide described abovecan further comprise one or more additional cargo peptides, attached toone another and ultimately to the carrier peptide.

Modifications to both the carrier and cargo have been made with thegoals of improving potency, stability in biological fluids/tissues, andchemical stability. These changes provide an εPKC inhibitor withenhanced properties for use in a variety of clinical indications.

Some of the modifications which have been applied include:

-   -   1. Capping the cargo and/or carrier peptides to hinder        proteolysis in vivo, and thereby to increase potency and/or        duration of efficacy;    -   2. Generating overlap peptides incorporating additional        contiguous regions of the parent protein to improve potency;    -   3. Making linear peptides which have cargo and carrier in a        single peptide chain to improve the chemical stability and        shelf-life of drug product;    -   4. Making multimer peptides which have two or more copies of the        active peptide to improve protease resistance and potency;    -   5. Making retro-inverso analogs of peptides to hinder        proteolysis; and    -   6. Introducing disulfide analogs to provide improved chemical        stability.

The modifications described herein improve the potency, plasmastability, and chemical stability of the modified εPKC inhibitorypeptides. Effective modifications to εPKC inhibitory peptides areidentified by selecting a prototype εPKC inhibitory peptide andmodifying these peptides to serve as cargo peptides for the treatment ofpain. The prototype peptide can be a presently known peptide or one asof yet unidentified as an εPKC inhibitory peptide. A preferred prototypesequence is E-A-V-S-L-K-P-T (SEQ ID NO:58), where the peptide isunmodified and conjugated to a carrier via Cys residues located at theamino termini of the cargo and carrier peptides, although any inhibitoryεPKC peptide can be used as the starting cargo sequence. A variety ofmodified or analog peptides are contemplated. Some such analogs compriseamino acid sequences that overlap and extend beyond the prototypesequence. Other analog peptides are truncated relative to the prototype.Additionally, analogs of the prototype sequence may have one or moreamino acid substitutions relative to the prototype sequence, wherein theamino acid substituted is an alanine residue or an aspartic acidresidue. The systematic generation of such alanine or aspartic acidcontaining peptides is known as “scanning.” The generation of linearpeptides comprising the analogs and modified carrier peptides is furthercontemplated.

Additional modifications to prototype sequences are directed atmodifying specific degradation sites within the cargo peptide orpeptides, the carrier peptide or peptides, or both, and introducingamino acid substitutions or other chemical modifications which blocksthese sites from degradation.

Table 1 lists a number of exemplary epsilon PKC inhibitory peptides foruse with the present invention as prototype sequences

TABLE 1 Peptides derived from epsilon PKC Peptide SEQ ID NO. SequencePosition V1-1 SEQ ID NO: 15 N-G-L-L-K-I-K PKC(5-11) V1-2 SEQ ID NO: 58E-A-V-S-L-K-P-T PKC(14-21) V1-3 SEQ ID NO: 16 L-A-V-F-H-D-A-P-I-G-YPKC(81-91) V1-4 SEQ ID NO: 17 D-D-F-V-A-N-C-T-I PKC(92-100) V1-5SEQ ID NO: 18 W-I-D-L-E-P-E-G-R-V PKC(116-125) V1-6 SEQ ID NO: 19H-A-V-G-P-R-P-Q-T-F PKC(27-36) V1-7 SEQ ID NO: 20 N-G-S-R-H-F-E-DPKC(108-115) V1-7.1 SEQ ID NO: 21 H-D-A-P-I-G-D-Y — V1-7.2 SEQ ID NO: 22H-D-A-P-I-G — V1-7.3 SEQ ID NO: 26 H-D-A-A-I-G-Y-D — V1-7.4SEQ ID NO: 27 H-D-A-P-I-P-Y-D — V1-7.5 SEQ ID NO: 28 H-N-A-P-I-G-Y-D —V1-7.6 SEQ ID NO: 29 H-A-A-P-I-G-Y-D — V1-7.7 SEQ ID NO: 30A-D-A-P-I-G-Y-D — V1-7.8 SEQ ID NO: 31 H-D-A-P-A-G-Y-D — V1-7.9SEQ ID NO: 32 H-D-A-P-I-G-A-D — V1-7.10 SEQ ID NO: 33 H-D-A-P-I-A-Y-D —V1-7.11 SEQ ID NO: 34 H-D-A-P-I-G-Y-A — V3-1 SEQ ID NO: 35S-S-P-S-E-E-D-R-S PKC(336-344) V3-2 SEQ ID NO: 36 P-C-D-Q-E-I-K-EPKC(351-358) V3-3 SEQ ID NO: 37 E-N-N-I-R-K-A-L-S PKC(360-368) V3-4SEQ ID NO: 38 G-E-V-R-Q-G-Q-A PKC(393-400) V5-1 SEQ ID NO: 39E-A-I-V-K-Q PKC(714-719) V5-2 SEQ ID NO: 40 I-K-T-K-R-D-V PKC(689-695)V5-2.1 SEQ ID NO: 41 I-K-T-K-R-L-I — V5-3 SEQ ID NO: 42 C-E-A-I-V-K-QPKC(714-719) V5-4 SEQ ID NO: 43 T-K-R-D-V-N-N-F-D-Q PKC(791-800)

As discussed more fully below, it is preferable that the εPKC inhibitorypeptide be chemically associated with a carrier moiety, such as acarrier peptide. In one embodiment, the inhibitory peptide and thecarrier peptide are linked via a disulfide bond. Electrostatic andhydrophobic interactions can also be exploited to associate chemicallythe carrier moiety with the εPKC inhibitory peptide. In the case of theforming a disulfide bond, it may be advantageous to add a Cys residue tothe PKC inhibitory peptide sequence or to the carrier peptide sequence.The Cys residue can be added to the amino or carboxy termini, or both.The Cys residue can also be located within the amino acid sequence ofthe cargo or carrier peptides. Such endogenous Cys residues have beenshown to stabilize a disulfide bond linkage between the carrier andcargo peptides. Another linking system involves linearizing peptides ofinterest using glycine residue linkers. One preferred embodiment isKP-1678, which has the sequence of εV1-2 and a TAT carrier peptide,wherein the amino terminus is acetylated and the carboxy terminus ismodified with an amino group (Ac-EAVSLKPTGGYGRKKRRQRRR-NH₂) (SEQ IDNO:57).

Hybrid Peptide Constructs

As discussed above a variety of modifications to PKC modulatory peptidesare contemplated. One example of such a modification includes theconstruction of linear peptides comprising, for example a cargo PKCmodulatory peptide and a carrier peptide. Another example is a multimerpeptide construct comprising a plurality of PKC modulatory peptides anda carrier peptide. Either peptide design model can be modified such thatmultiple modulatory PKC peptides are included, and those modulatorypeptides can be selected such that different PKC isozymes can bemodulated with the same construct.

The hybrid peptide approach has various advantages over a singlefunctionality peptide construct. For example, using multiple PKCmodulatory peptides in the same construct permits one to modulatesimultaneously two or more different PKC isozymes using otherwiseisozyme specific modulatory peptides. Joint modulation in this manner,while less specific than the use of isozyme specific peptides alone, isstill more specific than using isozyme non-specific peptide modulatorsand other small molecule type kinase inhibitors. The use of isozymespecific modulatory peptides is merely exemplary. Hybrid peptideconstructs can also comprise modulatory peptides which are eitherspecific or non-specific for any PKC isozyme.

Peptide modulators of any of the PKC isozymes are contemplated for useto construct hybrid peptide constructs. The PKC family of isozymesincludes at least 11 different protein kinases that can be divided intoat least three subfamilies based on their homology and sensitivity toactivators. Each isozyme includes a number of homologous (“conserved” or“C”) domains interspersed with isozyme-unique (“variable” or “V”)domains. Members of the “classical” or “cPKC” subfamily, α, β_(I),β_(II), and γPKC, contain four homologous domains (C1, C2, C3 and C4)and require calcium, phosphatidylserine, and diacylglycerol or phorbolesters for activation. In members of the “novel” or “nPKC” subfamily, δ,ε, η and εPKC, a C2-like domain precedes the C1 domain. However, that C2domain does not bind calcium and therefore the nPKC subfamily does notrequire calcium for activation. Finally, members of the “atypical” or“aPKC” subfamily, ζ and λPKC, lack both the C2 and one-half of the C1homologous domains and are insensitive to diacylglycerol, phorbol estersand calcium. Modulatory peptides with activity against one or more ofthe PKC isozymes can be used to prepare hybrid peptide constructs.

In a preferred embodiment, hybrid peptide constructs comprising one ormore εPKC modulatory peptides and one or more γPKC modulatory peptides.One a preferred embodiment, one or more εPKC inhibitory peptides areused with one or more γPKC inhibitory peptides to construct an εPKC-γPKChybrid inhibitory peptide.

Various εPKC inhibitory peptides are discussed above, and these peptidesare examples of some of the peptides that can be used to prepare hybridpeptide constructs. Various γPKC inhibitors are described herein and canbe used with the presently disclosed methods. The inhibitory peptide canbe derived from any domain, whether variable or constant. Thus,inhibitory peptides can be derived from V1, V2, V3, V4, or V5.Inhibitory peptides can also be derived from the constant regions C1(C1a, C1b), C3, C4, or C5. Peptides overlapping one or more of theseregions are also contemplated. The cargo peptides derived from thevarious domains and range in length from 5 to 30 amino acids in length.More particularly, the peptides derived from the PKC domain are 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 residues in length. Another source of prototypepeptides can be found in U.S. patent application Ser. No. 11/011,557,entitled, “Isozyme-specific antagonists of protein kinase C,” whichtakes activator peptides and converts them to inhibitor peptides, andwhich is hereby incorporated by reference in its entirety. A preferredγPKC inhibitory peptide prototype sequence is R-L-V-L-A-S (SEQ ID NO:1).All of the modifications described above in relation to the PKC peptidesare equally applicable to the γPKC inhibitory peptides contemplated foruse in the hybrid constructs.

The following tables list a number of exemplary gamma PKC inhibitorypeptides for use with the present invention as prototype sequences.

TABLE 2 BASIC SET CARGO CARRIER SEQ SEQ ID LINKER ID N-term Cargo NO:C-term Linker N-term Carrier NO: C-term Amine CRLVLAS 2 CarboxylDisulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl CRLVLAS 2 CarboxylDisulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl CRLVLAS 2 Amide DisulfideAmine CYGRKKRRQRRR 4 Carboxyl Amine CRLVLAS 2 Amide Disulfide AmineCYGRKKRRQRRR 4 Carboxyl Amine CRLVLAS 2 Carboxyl Disulfide AcetylCYGRKKRRQRRR 4 Carboxyl Amine CRLVLAS 2 Carboxyl Disulfide AcetylCYGRKKRRQRRR 4 Amide Amine CRLVLAS 2 Carboxyl Disulfide AmineCYGRKKRRQRRR 4 Amide Acetyl CRLVLAS 2 Amide Disulfide AcetylCYGRKKRRQRRR 4 Amide Amine RLVLASC 3 Carboxyl Disulfide AmineCYGRKKRRQRRR 4 Carboxyl Acetyl RLVLASC 3 Carboxyl Disulfide AmineCYGRKKRRQRRR 4 Carboxyl Acetyl RLVLASC 3 Amide Disulfide AmineCYGRKKRRQRRR 4 Carboxyl Amine RLVLASC 3 Amide Disulfide AmineCYGRKKRRQRRR 4 Carboxyl Amine RLVLASC 3 Carboxyl Disulfide AcetylCYGRKKRRQRRR 4 Carboxyl Amine RLVLASC 3 Carboxyl Disulfide AcetylCYGRKKRRQRRR 4 Amide Amine RLVLASC 3 Carboxyl Disulfide AmineCYGRKKRRQRRR 4 Amide Acetyl RLVLASC 3 Amide Disulfide AcetylCYGRKKRRQRRR 4 Amide Amine RLVLASC 3 Carboxyl Disulfide AmineYGRKKRRQRRRC 5 Carboxyl Acetyl RLVLASC 3 Carboxyl Disulfide AmineYGRKKRRQRRRC 5 Carboxyl Acetyl RLVLASC 3 Amide Disulfide AmineYGRKKRRQRRRC 5 Carboxyl Amine RLVLASC 3 Amide Disulfide AmineYGRKKRRQRRRC 5 Carboxyl Amine RLVLASC 3 Carboxyl Disulfide AcetylYGRKKRRQRRRC 5 Carboxyl Amine RLVLASC 3 Carboxyl Disulfide AcetylYGRKKRRQRRRC 5 Amide Amine RLVLASC 3 Carboxyl Disulfide AmineYGRKKRRQRRRC 5 Amide Acetyl RLVLASC 3 Amide Disulfide AcetylYGRKKRRQRRRC 5 Amide Amine CRLVLAS 2 Carboxyl Disulfide AmineYGRKKRRQRRRC 5 Carboxyl Acetyl CRLVLAS 2 Carboxyl Disulfide AmineYGRKKRRQRRRC 5 Carboxyl Acetyl CRLVLAS 2 Amide Disulfide AmineYGRKKRRQRRRC 5 Carboxyl Amine CRLVLAS 3 Amide Disulfide AmineYGRKKRRQRRRC 5 Carboxyl Amine CRLVLAS 2 Carboxyl Disulfide AcetylYGRKKRRQRRRC 5 Carboxyl Amine CRLVLAS 2 Carboxyl Disulfide AcetylYGRKKRRQRRRC 5 Amide Amine CRLVLAS 2 Carboxyl Disulfide AmineYGRKKRRQRRRC 5 Amide Acetyl CRLVLAS 2 Amide Disulfide AcetylYGRKKRRQRRRC 5 Amide

TABLE 3 HOMOCYSTEINE (homoC) CARGO CARRIER SEQ SEQ N- ID LINKER ID termCargo NO: C-term Linker N-term Carrier NO: C-term Amine homoC-RLVLAS 6Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl AcetylhomoC-RLVLAS 6 Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 CarboxylAcetyl homoC-RLVLAS 6 Amide Disulfide Amine homoC-YGRKKRRQRRR 8 CarboxylAmine homoC-RLVLAS 6 Amide Disulfide Amine homoC-YGRKKRRQRRR 8 CarboxylAmine homoC-RLVLAS 6 Carboxyl Disulfide Acetyl homoC-YGRKKRRQRRR 8Carboxyl Amine homoC-RLVLAS 6 Carboxyl Disulfide AcetylhomoC-YGRKKRRQRRR 8 Amide Amine homoC-RLVLAS 6 Carboxyl Disulfide AminehomoC-YGRKKRRQRRR 8 Amide Acetyl homoC-RLVLAS 6 Amide Disulfide AcetylhomoC-YGRKKRRQRRR 8 Amide Amine RLVLAS-homoC 7 Carboxyl Disulfide AminehomoC-YGRKKRRQRRR 8 Carboxyl Acetyl RLVLAS-homoC 7 Carboxyl DisulfideAmine homoC-YGRKKRRQRRR 8 Carboxyl Acetyl RLVLAS-homoC 7 Amide DisulfideAmine homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLAS-homoC 7 Amide DisulfideAmine homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLAS-homoC 7 CarboxylDisulfide Acetyl homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLAS-homoC 7Carboxyl Disulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine RLVLAS-homoC 7Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Amide Acetyl RLVLAS-homoC 7Amide Disulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine RLVLAS-homoC 7Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl AcetylRLVLAS-homoC 7 Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9 CarboxylAcetyl RLVLAS-homoC 7 Amide Disulfide Amine YGRKKRRQRRR-homoC 9 CarboxylAmine RLVLAS-homoC 7 Amide Disulfide Amine YGRKKRRQRRR-homoC 9 CarboxylAmine RLVLAS-homoC 7 Carboxyl Disulfide Acetyl YGRKKRRQRRR-homoC 9Carboxyl Amine RLVLAS-homoC 7 Carboxyl Disulfide AcetylYGRKKRRQRRR-homoC 9 Amide Amine RLVLAS-homoC 7 Carboxyl Disulfide AmineYGRKKRRQRRR-homoC 9 Amide Acetyl RLVLAS-homoC 7 Amide Disulfide AcetylYGRKKRRQRRR-homoC 9 Amide Amine homoC-RLVLAS 6 Carboxyl Disulfide AmineYGRKKRRQRRR-homoC 9 Carboxyl Acetyl homoC-RLVLAS 6 Carboxyl DisulfideAmine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl homoC-RLVLAS 6 Amide DisulfideAmine YGRKKRRQRRR-homoC 9 Carboxyl Amine homoC-RLVLAS 6 Amide DisulfideAmine YGRKKRRQRRR-homoC 9 Carboxyl Amine homoC-RLVLAS 6 CarboxylDisulfide Acetyl YGRKKRRQRRR-homoC 9 Carboxyl Amine homoC-RLVLAS 6Carboxyl Disulfide Acetyl YGRKKRRQRRR-homoC 9 Amide Amine homoC-RLVLAS 6Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9 Amide Acetyl homoC-RLVLAS 6Amide Disulfide Acetyl YGRKKRRQRRR-homoC 9 Amide

TABLE 4 HOMOCYSTEINE (homoC) — Cargo only CARGO CARRIER SEQ SEQ IDLINKER ID N-term Cargo NO: C-term Linker N-term Carrier NO: C-term AminehomoC-RLVLAS 6 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl AcetylhomoC-RLVLAS 6 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl AcetylhomoC-RLVLAS 6 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl AminehomoC-RLVLAS 6 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl AminehomoC-RLVLAS 6 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Carboxyl AminehomoC-RLVLAS 6 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Amide AminehomoC-RLVLAS 6 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Amide AcetylhomoC-RLVLAS 6 Amide Disulfide Acetyl CYGRKKRRQRRR 4 Amide AmineRLVLAS-homoC 7 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl AcetylRLVLAS-homoC 7 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl AcetylRLVLAS-homoC 7 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl AmineRLVLAS-homoC 7 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl AmineRLVLAS-homoC 7 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Carboxyl AmineRLVLAS-homoC 7 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Amide AmineRLVLAS-homoC 7 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Amide AcetylRLVLAS-homoC 7 Amide Disulfide Acetyl CYGRKKRRQRRR 4 Amide AmineRLVLAS-homoC 7 Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Carboxyl AcetylRLVLAS-homoC 7 Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Carboxyl AcetylRLVLAS-homoC 7 Amide Disulfide Amine YGRKKRRQRRRC 5 Carboxyl AmineRLVLAS-homoC 7 Amide Disulfide Amine YGRKKRRQRRRC 5 Carboxyl AmineRLVLAS-homoC 7 Carboxyl Disulfide Acetyl YGRKKRRQRRRC 5 Carboxyl AmineRLVLAS-homoC 7 Carboxyl Disulfide Acetyl YGRKKRRQRRRC 5 Amide AmineRLVLAS-homoC 7 Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Amide AcetylRLVLAS-homoC 7 Amide Disulfide Acetyl YGRKKRRQRRRC 5 Amide AminehomoC-RLVLAS 6 Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Carboxyl AcetylhomoC-RLVLAS 6 Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Carboxyl AcetylhomoC-RLVLAS 6 Amide Disulfide Amine YGRKKRRQRRRC 5 Carboxyl AminehomoC-RLVLAS 6 Amide Disulfide Amine YGRKKRRQRRRC 5 Carboxyl AminehomoC-RLVLAS 6 Carboxyl Disulfide Acetyl YGRKKRRQRRRC 5 Carboxyl AminehomoC-RLVLAS 6 Carboxyl Disulfide Acetyl YGRKKRRQRRRC 5 Amide AminehomoC-RLVLAS 6 Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Amide AcetylhomoC-RLVLAS 6 Amide Disulfide Acetyl YGRKKRRQRRRC 5 Amide

TABLE 5 HOMOCYSTEINE (homoC) — Carrier only CARGO CARRIER SEQ SEQ IDLINKER ID N-term Cargo NO: C-term Linker N-term Carrier NO: C-term AmineCRLVLAS 2 Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl AcetylCRLVLAS 2 Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl AcetylCRLVLAS 2 Amide Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl AmineCRLVLAS 2 Amide Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl AmineCRLVLAS 2 Carboxyl Disulfide Acetyl homoC-YGRKKRRQRRR 8 Carboxyl AmineCRLVLAS 2 Carboxyl Disulfide Acetyl homoC-YGRKKRRQRRR 8 Amide AmineCRLVLAS 2 Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Amide AcetylCRLVLAS 2 Amide Disulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine RLVLASC3 Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl Acetyl RLVLASC 3Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl Acetyl RLVLASC 3Amide Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLASC 3 AmideDisulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLASC 3 CarboxylDisulfide Acetyl homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLASC 3 CarboxylDisulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine RLVLASC 3 CarboxylDisulfide Amine homoC-YGRKKRRQRRR 8 Amide Acetyl RLVLASC 3 AmideDisulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine RLVLASC 3 CarboxylDisulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl RLVLASC 3 CarboxylDisulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl RLVLASC 3 AmideDisulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Amine RLVLASC 3 AmideDisulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Amine RLVLASC 3 CarboxylDisulfide Acetyl YGRKKRRQRRR-homoC 9 Carboxyl Amine RLVLASC 3 CarboxylDisulfide Acetyl YGRKKRRQRRR-homoC 9 Amide Amine RLVLASC 3 CarboxylDisulfide Amine YGRKKRRQRRR-homoC 9 Amide Acetyl RLVLASC 3 AmideDisulfide Acetyl YGRKKRRQRRR-homoC 9 Amide Amine CRLVLAS 2 CarboxylDisulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl CRLVLAS 2 CarboxylDisulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl CRLVLAS 2 AmideDisulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Amine CRLVLAS 2 AmideDisulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Amine CRLVLAS 2 CarboxylDisulfide Acetyl YGRKKRRQRRR-homoC 9 Carboxyl Amine CRLVLAS 2 CarboxylDisulfide Acetyl YGRKKRRQRRR-homoC 9 Amide Amine CRLVLAS 2 CarboxylDisulfide Amine YGRKKRRQRRR-homoC 9 Amide Acetyl CRLVLAS 2 AmideDisulfide Acetyl YGRKKRRQRRR-homoC 9 Amide

TABLE 6 MERCAPTOPROPIONIC ACID (MerPC) CARGO CARRIER SEQ SEQ ID LINKERID N-term Cargo NO: C-term Linker N-term Carrier NO: C-term AmineMerPC-RLVLAS 10 Carboxyl Disulfide Amine MerPC-YGRKKRRQRRR 11 CarboxylAcetyl MerPC-RLVLAS 10 Carboxyl Disulfide Amine MerPC-YGRKKRRQRRR 11Carboxyl Acetyl MerPC-RLVLAS 10 Amide Disulfide Amine MerPC-YGRKKRRQRRR11 Carboxyl Amine MerPC-RLVLAS 10 Amide Disulfide AmineMerPC-YGRKKRRQRRR 11 Carboxyl Amine MerPC-RLVLAS 10 Carboxyl DisulfideAcetyl MerPC-YGRKKRRQRRR 11 Carboxyl Amine MerPC-RLVLAS 10 CarboxylDisulfide Acetyl MerPC-YGRKKRRQRRR 11 Amide Amine MerPC-RLVLAS 10Carboxyl Disulfide Amine MerPC-YGRKKRRQRRR 11 Amide Acetyl MerPC-RLVLAS10 Amide Disulfide Acetyl MerPC-YGRKKRRQRRR 11 Amide Amine MerPC-RLVLAS10 Carboxyl Disulfide Amine CYGRKKRRQRRR  4 iCarboxyl AcetylMerPC-RLVLAS 10 Carboxyl Disulfide Amine CYGRKKRRQRRR  4 Carboxyl AcetylMerPC-RLVLAS 10 Amide Disulfide Amine CYGRKKRRQRRR  4 Carboxyl AmineMerPC-RLVLAS 10 Amide Disulfide Amine CYGRKKRRQRRR  4 Carboxyl AmineMerPC-RLVLAS 10 Carboxyl Disulfide Acetyl CYGRKKRRQRRR  4 Carboxyl AmineMerPC-RLVLAS 10 Carboxyl Disulfide Acetyl CYGRKKRRQRRR  4 Amide AmineMerPC-RLVLAS 10 Carboxyl Disulfide Amine CYGRKKRRQRRR  4 Amide AcetylMerPC-RLVLAS 10 Amide Disulfide Acetyl CYGRKKRRQRRR  4 Amide AmineCRLVLAS  2 Carboxyl Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl AcetylCRLVLAS  2 Carboxyl Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl AcetylCRLVLAS  2 Amide Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl AmineCRLVLAS  2 Amide Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl AmineCRLVLAS  2 Carboxyl Disulfide Acetyl MerPC-YGRKKRRQRRR 11 Carboxyl AmineCRLVLAS  2 Carboxyl Disulfide Acetyl MerPC-YGRKKRRQRRR 11 Amide AmineCRLVLAS  2 Carboxyl  Disulfide Amine MerPC-YGRKKRRQRRR 11 Amide AcetylCRLVLAS  2 Amide Disulfide Acetyl MerPC-YGRKKRRQRRR 11 Amide

TABLE 7 MERCAPTOACETIC ACID (MerAC) CARGO CARRIER SEQ SEQ ID LINKER IDN-term Cargo NO: C-term Linker N-term Carrier NO: C-term AmineMerAC-RLVLAS 24 Carboxyl Disulfide Amine MerAC- 25 Carboxyl YGRKKRRQRRRAcetyl MerAC-RLVLAS 24 Carboxyl Disulfide Amine MerAC- 25 CarboxylYGRKKRRQRRR Acetyl MerAC-RLVLAS 24 Amide Disulfide Amine MerAC- 25Carboxyl YGRKKRRQRRR Amine MerAC-RLVLAS 24 Amide Disulfide Amine MerAC-25 Carboxyl YGRKKRRQRRR Amine MerAC-RLVLAS 24 Carboxyl Disulfide AcetylMerAC- 25 Carboxyl YGRKKRRQRRR Amine MerAC-RLVLAS 24 Carboxyl DisulfideAcetyl MerAC- 25 Amide YGRKKRRQRRR Amine MerAC-RLVLAS 24 CarboxylDisulfide Amine MerAC- 25 Amide YGRKKRRQRRR Acetyl MerAC-RLVLAS 24 AmideDisulfide Acetyl MerAC- 25 Amide YGRKKRRQRRR Amine MerAC-RLVLAS 24Carboxyl Disulfide Amine CYGRKKRRQRRR  4 Carboxyl Acetyl MerAC-RLVLAS 24Carboxyl Disulfide Amine CYGRKKRRQRRR  4 Carboxyl Acetyl MerAC-RLVLAS 24Amide Disulfide Amine CYGRKKRRQRRR  4 Carboxyl Amine MerAC-RLVLAS 24Amide Disulfide Amine CYGRKKRRQRRR  4 Carboxyl Amine MerAC-RLVLAS 24Carboxyl Disulfide Acetyl CYGRKKRRQRRR  4 Carboxyl Amine MerAC-RLVLAS 24Carboxyl Disulfide Acetyl CYGRKKRRQRRR  4 Amide Amine MerAC-RLVLAS 24Carboxyl Disulfide Amine CYGRKKRRQRRR  4 Amide Acetyl MerAC-RLVLAS 24Amide Disulfide Acetyl CYGRKKRRQRRR  4 Amide Amine CRLVLAS  2 CarboxylDisulfide Amine MerAC- 25 Carboxyl YGRKKRRQRRR Acetyl CRLVLAS  2Carboxyl Disulfide Amine MerAC- 25 Carboxyl YGRKKRRQRRR Acetyl CRLVLAS 2 Amide Disulfide Amine MerAC- 25 Carboxyl YGRKKRRQRRR Amine CRLVLAS  2Amide Disulfide Amine MerAC- 25 Carboxyl YGRKKRRQRRR Amine CRLVLAS  2Carboxyl Disulfide Acetyl MerAC- 25 Carboxyl YGRKKRRQRRR Amine CRLVLAS 2 Carboxyl Disulfide Acetyl MerAC- 25 Amide YGRKKRRQRRR Amine CRLVLAS 2 Carboxyl Disulfide Amine MerAC- 25 Amide YGRKKRRQRRR Acetyl CRLVLAS 2 Amide Disulfide Acetyl MerAC- 25 Amide YGRKKRRQRRR

TABLE 8 MERCAPTOBUTYRIC ACID (MerBC) CARGO CARRIER SEQ SEQ N- ID LINKERID term Cargo NO: C-term Linker N-term Carrier NO: C-term AmineMerBC-RLVLAS 23  Carboxyl Disulfide Amine MerBC- 12 Carboxyl YGRKKRRQRRRAcetyl MerBC-RLVLAS 23  Carboxyl Disulfide Amine MerBC- 12 CarboxylYGRKKRRQRRR Acetyl MerBC-RLVLAS 23 Amide Disulfide Amine MerBC- 12Carboxyl YGRKKRRQRRR Amine MerBC-RLVLAS 23 Amide Disulfide Amine MerBC-12 Carboxyl YGRKKRRQRRR Amine MerBC-RLVLAS 23 Carboxyl Disulfide AcetylMerBC- 12 Carboxyl YGRKKRRQRRR Amine MerBC-RLVLAS 23 Carboxyl DisulfideAcetyl MerBC- 12 Amide YGRKKRRQRRR Amine MerBC-RLVLAS 23 CarboxylDisulfide Amine MerBC- 12 Amide YGRKKRRQRRR Acetyl MerBC-RLVLAS 23 AmideDisulfide Acetyl MerBC- 12 Amide YGRKKRRQRRR Amine MerBC-RLVLAS 23Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl MerBC-RLVLAS 23Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl MerBC-RLVLAS 23Amide Disulfide Amine  CYGRKKRRQRRR 4 Carboxyl Amine MerBC-RLVLAS 23Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine MerBC-RLVLAS 23Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Carboxyl Amine MerBC-RLVLAS 23Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine MerBC-RLVLAS 23Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Amide Acetyl MerBC-RLVLAS 23Amide Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine CRLVLAS  2 CarboxylDisulfide Amine MerBC- 12 Carboxyl YGRKKRRQRRR Acetyl CRLVLAS  2Carboxyl Disulfide Amine MerBC- 12 Carboxyl YGRKKRRQRRR Acetyl CRLVLAS 2 Amide Disulfide Amine MerBC- 12 Carboxyl YGRKKRRQRRR Amine CRLVLAS  2Amide Disulfide Amine MerBC- 12 Carboxyl YGRKKRRQRRR Amine CRLVLAS  2Carboxyl Disulfide Acetyl MerBC- 12 Carboxyl YGRKKRRQRRR Amine CRLVLAS 2 Carboxyl Disulfide Acetyl MerBC- 12 Amide YGRKKRRQRRR Amine CRLVLAS 2 Carboxyl Disulfide Amine MerBC- 12 Amide YGRKKRRQRRR Acetyl CRLVLAS 2 Amide Disulfide Acetyl MerBC- 12 Amide YGRKKRRQRRR

TABLE 9 Ala-Cys CARGO CARRIER SEQ SEQ ID LINKER ID N-term Cargo NO:C-term Linker N-term Carrier NO: C-term Amine ACRLVLAS 13 CarboxylDisulfide Amine ACYGRKKRRQRRR 14 Carboxyl Acetyl ACRLVLAS 13 CarboxylDisulfide Amine ACYGRKKRRQRRR 14 Carboxyl Acetyl ACRLVLAS 13 AmideDisulfide Amine ACYGRKKRRQRRR 14 Carboxyl Amine ACRLVLAS 13 AmideDisulfide Amine ACYGRKKRRQRRR 14 Carboxyl Amine ACRLVLAS 13 CarboxylDisulfide Acetyl ACYGRKKRRQRRR 14 Carboxyl Amine ACRLVLAS 13 CarboxylDisulfide Acetyl ACYGRKKRRQRRR 14 Amide Amine ACRLVLAS 13 CarboxylDisulfide Amine ACYGRKKRRQRRR 14 Amide Acetyl ACRLVLAS 13 AmideDisulfide Acetyl ACYGRKKRRQRRR 14 Amide Amine ACRLVLAS 13 CarboxylDisulfide Amine CYGRKKRRQRRR  4 Carboxyl Acetyl ACRLVLAS 13 CarboxylDisulfide Amine CYGRKKRRQRRR  4 Carboxyl Acetyl ACRLVLAS 13 AmideDisulfide Amine CYGRKKRRQRRR  4 Carboxyl Amine ACRLVLAS 13 AmideDisulfide Amine CYGRKKRRQRRR  4 Carboxyl Amine ACRLVLAS 13 CarboxylDisulfide Acetyl CYGRKKRRQRRR  4 Carboxyl Amine ACRLVLAS 13 CarboxylDisulfide Acetyl CYGRKKRRQRRR  4 Amide Amine ACRLVLAS 13 CarboxylDisulfide Amine CYGRKKRRQRRR  4 Amide Acetyl ACRLVLAS 13 Amide DisulfideAcetyl CYGRKKRRQRRR  4 Amide Amine CRLVLAS  2 Carboxyl Disulfide AmineACYGRKKRRQRRR 14 Carboxyl Acetyl CRLVLAS  2 Carboxyl Disulfide AmineACYGRKKRRQRRR 14 Carboxyl Acetyl CRLVLAS  2 Amide Disulfide AmineACYGRKKRRQRRR 14 Carboxyl Amine CRLVLAS  2 Amide Disulfide AmineACYGRKKRRQRRR 14 Carboxyl Amine CRLVLAS  2 Carboxyl Disulfide AcetylACYGRKKRRQRRR 14 Carboxyl Amine CRLVLAS  2 Carboxyl Disulfide AcetylACYGRKKRRQRRR 14 Amide Amine CRLVLAS  2 Carboxyl Disulfide AmineACYGRKKRRQRRR 14 Amide Acetyl CRLVLAS  2 Amide Disulfide AcetylACYGRKKRRQRRR 14 Amide

All of the PKC peptides discussed in U.S. Provisional No. 60/910,588,filed Apr. 6, 2007 are hereby incorporated by reference.

Carrier Moiety

A wide variety of molecules (particularly macromolecules such aspeptides) intended for cellular uptake have been found to be poorlytransported across cell membranes. Among the solutions proposed tofacilitate cellular uptake have been the use of carrier moieties such ascationic (i.e., positively charged) polymers, peptides and antibodysequences, including polylysine, polyarginine, Antennapedia-derivedpeptides, HIV Tat-derived peptides and the like. (See, for example, U.S.Pat. Nos. 4,847,240, 5,888,762, 5,747,641, 6,593,292, US2003/0104622,US2003/0199677 and US2003/0206900.)

A particular example of a cargo/carrier conjugate is KP-1634 (SEQ IDNO:49), which is made up of two εPKC-derived peptides with aminoterminal caps and a HIV Tat-derived carrier peptide which has beencapped at both its amino and carboxy terminal ends.

Additional Inhibitors

Additional inhibitors of εPKC can be identified using assays thatmeasure the activation, intracellular translocation, binding tointracellular receptors (e.g. RACKs) or catalytic activity of εPKC.Traditionally, the kinase activity of PKC family members has beenassayed using at least partially purified PKC in a reconstitutedphospholipid environment with radioactive ATP as the phosphate donor anda histone protein or a short peptide as the substrate (T. Kitano, M. Go,U. Kikkawa, Y. Nishizuka, Meth. Enzymol. 124, 349-352 (1986); R. O.Messing, P. J. Peterson, C. J. Henrich, J. Biol. Chem. 266, 23428-23432(1991)). Recent improvements include a rapid, highly sensitivechemiluminescent assay that measures protein kinase activity atphysiological concentrations and can be automated and/or used inhigh-throughput screening (C. Lehel, S. Daniel-Issakani, M. Brasseur, B.Strulovici, Anal. Biochem. 244, 340-346 (1997)) and an assay using PKCin isolated membranes and a selective peptide substrate that is derivedfrom the MARCKS protein (B. R. Chakravarthy, A Bussey, J. F. Whitfield,M. Sikorska, R. E. Williams, J. P. Durkin, Anal. Biochem. 196, 144-150(1991)). Inhibitors that affect the intracellular translocation of εPKCcan be identified by assays in which the intracellular localization ofεPKC is determined by fractionation (R. O. Messing, P. J. Peterson, C.J. Henrich, J. Biol. Chem. 266, 23428-23432 (1991)) orimmunohistochemistry (U.S. Pat. No. 5,783,405; U.S. patent applicationSer. No. 08/686,796 now U.S. Pat. No. 6,255,057, now U.S. Pat. No.6,255,057). To identify an inhibitor of εPKC, the assays should beperformed with εPKC. The selectivity of such εPKC inhibitors can bedetermined by comparing the effect of the inhibitor on εPKC with itseffect on other PKC isozymes. The relevant portions of foregoing patentsand publications are hereby incorporated by reference.

Additional assays for identifying sPKC inhibitors can be found in U.S.Pat. Nos. 5,783,405, 6,156,977, and 6,423,684, all of which are herebyincorporated by reference in their entirety.

Mechanisms

Experimental data reported here shows that (1) locally-delivered εPKCinhibitory peptides to the hindlimbs of test animals producedanti-hyperalgesic/analgesic effects in neuropathetic and inflammatorypain models; (2) such effects of the εPKC inhibitory peptides are thoughto be mediated by nerves traveling inside the sciatic and saphenousafferents, which project to the hindlimbs and thus could be part of aneuronal reflex subject to be inhibited by the εPKC inhibitory peptides;(3) the effects of the εPKC inhibitory peptides appears to besympathoadrenal-dependent, which coincides with the reports that εPKCnot only exists in sympathetic ganglia and functions to theneurotransmitter-releasing process (e.g., Scholze et al.: J. Neurosci.,22: 5823-32, 2002) but also modulates sensitization of the C-fibers (apain-sensing nerve) (Khasar, et al., Neuron, 24: 253-260, 1999); and (4)the anti-hyperalgesic/analgesic effects of the εPKC inhibitory peptidesis mediated by adrenoceptors. Of note, epinephrine (EPI), one of theneurotransmitters released from the adrenal medulla and sympatheticpost-ganglionic neuron (SPGN) terminals, is known to sensitize theC-fibers (Chen & Levine, J. Pain, 6: 439-446, 2005; Khasar, et al.,Neuron, 24: 253-260, 1999). It was reported that εPKC modulates Ca⁺⁺influx (Boehm et al., J. Neurosci., 16: 4516-603, 1996) byphosphorylating the N-type calcium channels (Zhu and Ikeda, J.Neurophysiol., 74: 1546-60, 1994) and thus regulates the release ofcatecholamines from the sympathetic neurons (Scholze et al.: J.Neurosci., 22: 5823-32, 2002).

Identification of the anti-hyperalgesic effects of εPKC inhibitorypeptides differs from that discussed by Messing & Levine (U.S. Pat. No.6,686,334 B2, U.S. Pat. No. 6,376,467 B1 and 2002/0151465 A1), in whichexogenous EPI or isopropranolol (ISO, a synthetic β-adrenoceptoragonist) acts on the C-fibers via stimulating β-adrenoceptors. In thatwork, the pro-hyperalgesic effects of exogenous EPI or ISO areεPKC-dependent and do not act on the autoregulatory α₂-adrenoceptors onthe SPGN terminals. U.S. Pat. Nos. 6,686,334 and 6,376,467 and Pub. No.2002/0151465 A1 focused on the anti-hyperalgesic effects of εV_(1/2) onthe downstream, that is, the effects caused by exogenous catecholaminesor, in theory by endogenous catecholamines after they are released fromthe sympathoadrenal system. The presently disclosed data does notcontradict Messing & Levine, but rather focuses and extend theunderstanding the role εPKC plays in pain neurotransmission.Furthermore, the findings presented here demonstrate that theanti-hyperalgesic effects of the εPKC inhibitory peptides are alsomediated via “the upstream” neurotransmission in the SPGN, and morespecifically highlights the effects of the εPKC inhibitory peptides haveon the sympathoadrenal system.

This invention shows that administration of a εPKC inhibitor locally ata remote site can inhibit pain by a mechanism that is mediated via theSPGN, and is not limited by nor does it require systemic distribution ofthe inhibitor throughout the body. The remote action, rapid onset andlow doses of the εPKC inhibitor needed to inhibit the pain responserequire intact nerve since transection of the sciatic and saphenousnerves (de-afferentation) on the contralateral limb blocked the abilityof S.C administered εPKC to inhibit pain from a remote site.Furthermore, bilateral lumbar sympathectomy plus bilateral suprarenalganglionectomy as well as unilateral injection of phentolamine (which isa non-selective α-adrenoceptor antagonist) abolished the pain-reducingeffects of εPKC inhibitors.

The very low doses, rapid onset and remote action observed with εPKCinhibitor support the conclusion that perhaps even a much less selectiveinhibitor of εPKC (including inhibitors that might otherwise be toxic ifsystemically administered) could be used to suppress a pain response.Furthermore, since we have shown that very low doses of an εPKCadministered locally can have this effect, it is conceivable that asimilar very low dose of an otherwise non-selective and systemicallytoxic εPKC inhibitor could be administered locally at very low doses(i.e., much below the dose-limiting systemic toxic levels of that drug)to achieve inhibition of a pain response without producing any or onlyvery limited systemic toxic side effects.

Methods of Use and Formulations

The modified peptides described herein are useful for the prevention andtreatment of pain. For the purposes of this discussion, pain, and thetreatment thereof, is categorized into different classes: treatment ofacute, chronic, neuropathic, and inflammatory pain. The modified εPKCinhibitory peptides described herein are useful for the treatment ofacute, chronic, neuropathic, and inflammatory pain.

Interestingly, the compounds disclosed herein are also useful inattenuated or preventing the development of neuropathic pain caused by aplurality of stimuli. For example, as discussed in Example 7 below,chronic inflammatory pain can be induced by the administration ofcarrageenan followed by the administration of prostaglandin E2. Thisphenomenon serves as a model for a variety of systems where a subjectreceiving a plurality of pain stimuli or pain sensitizing agents resultsin chronic inflammatory or neuropathic pain. It has been noted thatchemotherapy patients receiving TAXOL develop neuropathic pain, whichtypically resolves after the initial dose or doses of the drug. However,chemotherapy patients receive a full course of TAXOL treatments are leftwith on-going neuropathic pain. The present disclosure contemplates thatthe administration of the εPKC inhibitory peptides described herein,either prophylactically, with the chemotherapeutic agent, or subsequentto the chemotherapy will be effective to attenuate or prevent thedevelopment of the chronic inflammatory or neuropathic pain condition.

Once a cargo/carrier peptide construct has been assembled and tested forincreased stability, potency, or both as compared to a prototype, theconstruct is placed into a pharmaceutically acceptable formulation foradministration to a subject prior to, during, or continuously through apain inducing event.

A “pharmaceutically acceptable formulation” comprises one that issuitable for administering the modified εPKC inhibitor in a manner thatgives the desired results and does not also produce adverse side effectssufficient to convince a physician that the potential harm to a patientis greater than the potential benefit to that patient. The components ofa suitable pharmaceutically acceptable formulation for use with amodified εPKC inhibitors are determined in part by the route and methodof administration. The formulations generally comprise one or moremodified εPKC inhibitory peptides incorporated into a pharmaceuticallyacceptable carrier typically comprising simple chemicals such as sugars,amino acids or electrolytes. Exemplary solutions are typically preparedwith saline or buffer. The pharmaceutically acceptable carrier maycontain excipients which are well known in the art, and may be used in avariety of formulations. See, e.g., Remington's Pharmaceutical Sciences,18th Edition, A. R. Gennaro, Editor, Mack Publishing Company (1990);Remington: The Science and Practice of Pharmacy, 20th Edition, A. R.Gennaro, Editor, Lippincott Williams & Wilkins (2000); Handbook ofPharmaceutical Excipients, 3rd Edition, A. H. Kibbe, Editor, AmericanPharmaceutical Association, and Pharmaceutical Press (2000); andHandbook of Pharmaceutical Additives, compiled by Michael and Irene Ash,Gower (1995).

Inhibitor dosage in the formulation will vary according to a variety ofparameters influenced by the stability and potency of the cargo/carrierconstruct, the route of administration, and desired dosing regime. Dailydosages in the range of 1 μg/kg-100 mg/kg of body weight, preferably 1μg/kg-1 mg/kg and most preferably 10 μg/kg-1 mg/kg are contemplated.

Modified εPKC inhibitors can be administered locally or systemically.Local administration can be achieved by topical administration,transdermal, intradermal administration, intrathecal administration,intraperitoneal administration, or subcutaneous injection. Systemicadministration of a modified εPKC inhibitor is preferably parenteral,although oral, buccal, and intranasal administration is alsocontemplated. Parenteral administration is generally characterized byinjection, either subcutaneously, intramuscularly, intraperitoneal, andintravenously. Injectable forms of the modified inhibitory peptides canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid (e.g., dried or lyophilized) forms suitable forreconstitution into solution or suspension in liquid prior to injection,or as emulsions. Generally, suitable excipients include, for example,water, saline, dextrose, glycerol, ethanol or the like. In addition,minor amounts of non-toxic auxiliary substances can be employed, such aswetting or emulsifying agents, pH buffering agents, solubilityenhancers, tonicifiers and the like including, for example, sodiumacetate, sorbitan monolaurate, triethanolamine oleate, cyclodextrins,etc.

The modified εPKC inhibitory peptides can be administered to treat painas necessary. For prophylaxis, the modified εPKC compound may beadministered prior to a pain-inducing event. For example, the peptidecan be administered 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes,one hour, several hours, one day, several days, one week, or weeks priorahead of an anticipated pain-inducing event. Even longer periods ofprophylactic administration can be achieved using modified peptides thatare particularly stable in vivo, or by using a sustained releaseformulation of the peptide, e.g. delivery by intrathecal pump.

EXAMPLES

The following examples serve to describe more fully the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

Example 1 Analogs of εPKC Inhibitor Optimization

A prior art εPKC inhibitory sequence (KP-1636) was used as the prototypeto study the impacts of various chemical modifications on potency andstability. The prototype sequence, KP-1586 was used as the template inthe work described below. Various cargo and carrier peptides weremodified for this work, and these are provided in Table 10.

TABLE 10 Compound Carrier Peptide Description Cargo Peptide SequencesDescription KP-1586     C-E-A-V-S-L-K-P-T Cap Tat KP-1630 Ac-C-E-A-V-S-L-K-P-T-NH₂ Cap Tat KP-1631  Ac-C-L-K-P-T-A-W-S-L-R-NH₂Cap Tat KP-1632  Ac-C-E-A-V-S-L-K-P-T-A-W-S-L-R Cap Tat KP-1633      E-A-V-S-L-K-P-T-G-G-TaT-NH₂ Linear KP-1634  [Ac-E-A-V-S-L-K-P-T-G-G]-K-C Cap Tat KP-1635     t-p-k-l-s-v-a-e-cCap Tat KP-1636     C-E-A-V-S-L-K-P-T Tat KP-1637 Ac-C-E-A-V-S-L-K-P-T-NH₂ Ac-hCys-Tat KP-1638 Ac-hC-E-A-V-S-L-K-P-T-NH₂Ac-hCys-Tat KP-1678 Ac-E-A-V-S-L-K-P-T-G-G-Tat-NH₂ Linear

“Tat” in Table 10 refers to the fragment 47-57 of HIV Tat, and “Cap Tat”refers to an N-acetyl or C-amide analog of the same peptide. The term“hC” or “hCys” refers to a homocysteine amino acid.

Example 2 Stability of εPKC Inhibitor Analogs

The plasma and chemical stability of various cargo/carrier peptideconstructs described in Example 1 was tested. Plasma stability of thecompounds was tested using both human and rat sera and the amount ofstarting material was determined after 30 minutes of treatment. Chemicalstability was evaluated by determining the amount of starting materialremaining after 9 days of treatment.

TABLE 11 Plasma Stability % Remaining at 30 min. Chemical StabilityCompound Human Rat % Remaining at 9 days 1586 Lead 85 77 57 1630 Capcargo/Tat 76 77 67 1631 Overlap 1 49 67 76 1632 Overlap 2 54 61 89 1633Linear 83 77 99 1634 Dimer/cTat 87 106 82 1635 retro-inverso 63 72 951636 no caps 79 79 27 1637 half-HomoCys 60 78 100 1638 full HomoCys ndnd 100

Example 3 Time Course Plasma Stability of εPKC Inhibitor Analogs

The plasma stability of various cargo/carrier peptide constructsdescribed in Example 1 was tested using both human and rat sera and theamount of starting material was determined over time to 30 minutes oftreatment. The time course data for cargo/carrier peptides KP-1586,KP-1630, and KP-1631 is shown in FIG. 3, the data for peptides KP-1632,KP-1633, and KP-1634 is shown in FIG. 4, and the data for peptidesKP-1635, KP-1636, and KP-1637 is shown in FIG. 5. The dimer peptide andthe analog containing an uncapped carrier peptide were more stable thatof the prototype material. Interestingly, capping of the cargo peptidehad little impact on plasma stability as seen when comparing thestability of KP-1586 and KP-1630.

Example 4 Time Course Chemical Stability of εPKC Inhibitor Analogs

The chemical stability of various cargo/carrier peptide constructsdescribed in Example 1 was tested by examining the relativeconcentration of the peptides over a time period of more than 200 hoursat 37° C. The time course data for the cargo/carrier peptides is shownin FIG. 6. The data from this study showed that the prototype sequencewas only moderately stable as compared to the analogs. Both the linearand homocysteine-containing constructs showed improved stabilityrelative to the prototype sequence. For example, KP-1637 showed markedstability.

Example 5 Attenuation of Acute Pain with a Modified εPKC InhibitoryPeptide

A formalin-induced pain test was used to study the ability of a modifiedεPKC peptide to attenuate acute pain. Formalin was administered by anintraplantar route to all rats used in the present study. Test subjectsreceived a formulation containing modified εPKC peptide KP-1586 byintrathecal administration 15 minutes prior to the pain-causing agent(prophylactic mode). Two different concentrations of the modifiedpeptide were used in the test subjects. The data from this experiment isshown in FIG. 7. The results of this study indicate that theprophylactic administration of the modified εPKC inhibitory peptide waseffective to reduce flinches per minute in the test animals. Thus, theadministration of modified εPKC peptides is effective to attenuate anacute pain stimulus.

Example 6 Attenuation of Chronic Pain with a Modified εPKC InhibitoryPeptide

The Chung (L5 nerve transection) is a well-known model for chronic(neuropathic) pain. A representative modified εPKC peptide KP-1586provided in the disclosure has been efficacious in reducing pain in thismodel when delivered systemically. The results from this work are shownin FIG. 8.

The test peptide KP-1586, but not the control peptide, suppressedthermal hyperalgesia in a modified Chung's model when delivered for manydays by subcutaneous osmotic pump. Such inhibitory effects weredose-dependent, with an initial dose of 10-50 pmol/day.Anti-hyperalgesic effects became detectable as early as the next dayafter implantation and continued throughout at least one week withcontinuous compound delivery.

In the same model, chronic sub-cutaneous delivery of KP-1586, but notthe control peptide KP-1587, suppressed mechanical allodynia. Thisinhibition was dose-dependent in some observation windows, particularlyin the tests of the 7th day following establishment of the model.Anti-allodynic effects became detectable as early as the day afterimplantation.

KP-1586 was further able to modify pain responses in the Chung modelfollowing intrathecal administration. The efficacy of the drug in thismode lasted at least 90 minutes following single bolus administration.

Example 7 Attenuation of Chronic Inflammatory Pain using a Modified εPKCInhibitory Peptide

The intraplantar administration of carrageenan followed 5 days laterwith prostaglandin E2 (PGE2) to the rat paw causes both acute andchronic pain through an inflammatory mechanism. As shown in FIG. 9,local delivery of compounds described herein is able to attenuate thedevelopment of the pain response.

Representative compound was KP-1586 was able to reverse the painfuleffects of carrageenan when delivered by intradermal administrationwhile the control peptide KP-1587 did not.

Example 8 Subcutaneous εPKC Inhibitor Reverses Inflammatory Pain

The intraplantar administration of carrageenan followed 1 hour laterwith a subcutaneous bolus administration of KP-1634 was tested in amechanical pain model to demonstrate the effect of εPKC inhibition onpaw withdrawal. As shown in FIG. 10, paw withdrawal thresholds increasedmarkedly after the administration of the inhibitor while the controlpeptide KP-1587 did not induce the same effect.

Example 9 Prevention of Neuropathic Pain with SubcutaneousAdministration of a εPKC Inhibitory Peptide

The εPKC peptide KP-1586 was used in the Chung model to test the abilityof εPKC inhibitory peptides administered subcutaneously to preventneuropathic pain. The test peptides were administered at 1, 10, 50, and100 pmol/day. The results from this work are shown in FIG. 11.

The test peptide KP-1586, but not the control peptide 1587, suppressedthermal hyperalgesia in a modified Chung's model when delivered bysubcutaneous osmotic pump and tested as 1, 3 and 5 days post-surgery.

Example 10 Reversal of Neuropathic Pain with Subcutaneous Administrationof a εPKC Inhibitory Peptide

The εPKC peptide KP-1586 was used in the Chung model to test the abilityof εPKC inhibitory peptides administered subcutaneously to reverseneuropathic pain. The test peptides were administered at 0.1, 1, 10, 50,1000 pmol/day. The results from this work are shown in FIG. 12. FIG. 13shows the effect of an inhibitor administered at 10 pmole per day bysubcutaneous infusion with the pump being implanted 7 days after thetransection event.

Example 11 The Role of Sympathetic Terminals in SuppressingCarrageenan-Induced Mechanical Hyperalgesia by Inhibitors of PKC Epsilonin Rats

λ-carrageenan (Carr)-induced inflammatory pain involves activity of thesympathetic nervous system. Since the sympathetic neurons, like thesensory neurons, are rich in the epsilon isozyme of protein kinase C(εPKC), and since εPKC inhibitors reduce Carr-induced mechanicalhyperalgesia, it is hypothesized that the mechanism of action for εPKCinhibitors requires the sympathetic post-ganglionic nerve (SPGN)terminals.

The nociceptive flexior reflex was quantified on lightly restrained ratsby using a Basile Analgesymeter (Randall-Selitto test). Inflammatoryhyperlagesia was induced by injection of Carr (1%, 5 uL, i.d.), whichwas injected unilaterally to the dorsum of the hindpaw one-hour prior tocompound administration. The εPKC inhibitor peptide (εV1-2) was injectedvia systemic routes.

Subcutaneous injection of εV1-2 dose-dependently suppressed Carr-inducedmechanical hyperalgesia, independent of the site of administration(ipsilateral or contralateral hindlimb or the dorsal trunk).

In contrast, when εV1-2 was administered subcutaneously distal to atransection of the sciatic and saphenous nerves, there was no longersuppression of Carr-induced mechanical hyperalgesia of the contralateralpaw. This result suggested that tonic neuronal signaling in these nervesmay be the site of action for εV1-2. Furthermore, surgical removal ofbilateral lumbar sympathetic chain (L2-L4) and bilateral suprarenalganglia 7-day prior to the experiment completely abolishedanti-hyperalgesic effects of εV1-2. The effect of the surgicalsympathectomy was mimicked by acute treatment with adrenergicantagonists such as phentolamine (10 ug, s.c. injection to thecontralateral limb, just before Carr injection).

Results from the current study suggest a mechanism of action for εPKCand demonstrate anti-hyperalgesic effects of systemically-deliveredεV1-2. These results support the potential development of εPKCinhibitors as novel therapies for inflammatory pain.

Example 12 Modulation of Pain Responses using Peptides for SelectiveModulation of Protein Kinase C

It is known that λ-carrageenan (Carr)-induced inflammatory pain involvesactivity of the sympathetic nervous system. Since the sympatheticneurons, like the sensory neurons, are rich in the ε isozyme of proteinkinase C (εPKC), and since εPKC inhibitors reduce Carr-inducedmechanical hyperalgesia, it is hypothesized that the mechanism of actionfor εPKC inhibitors requires the sympathetic post-ganglionic nerve(SPGN) terminals.

The nociceptive flexior reflex was quantified on lightly restrained ratsby using a Basile Analgesymeter (Randall-Selitto test). Inflammatoryhyperlagesia was induced by injection of Carr (1%, 5 μL, i.d.), whichwas injected unilaterally to the dorsum of the hindpaw one-hr prior tocompound administration. The εPKC inhibitor peptide (εV₁₋₂) was injectedvia systemic routes.

Subcutaneous injection of εV₁₋₂ dose-dependently suppressed Carr-inducedmechanical hyperalgesia, independent of the site of administration(ipsilateral or contralateral hindlimb or the dorsal trunk).

In contrast, when εV₁₋₂ was administered subcutaneously distal to atransaction of the sciatic and saphenous nerves, there was no longersuppression of Carr-induced mechanical hyperalgesia of the contralateralpaw. This result suggested that tonic neuronal signaling in these nervesmay be the site of action for εV₁₋₂. Furthermore, surgical removal ofbilateral lumbar sympathetic chain (L₂-L₄) and bilateral suprarenalganglia 7-day prior to the experiment completely abolishedanti-hyperalgesic effects of εV₁₋₂. The effect of the surgicalsympathectomy was mimicked by acute treatment with adrenergicantagonists such as phentolamine (10 ug, s.c. injection to thecontralateral limb, just before Carr injection). See FIGS. 14-17.

Example 12 Nonclinical Pharmacology of KAI-1678

The ability of KAI-1678 to reduce allodynia, the heightened response tonormally innocuous stimuli, and hyperalgesia, the heightened response topainful stimuli, has been evaluated in rat models of inflammatory pain(carrageenan-induced pain) and neuropathic pain (L5 spinal nervetransection). In the carrageenan-induced inflammatory pain model, localintradermal administration of KAI-1678 has been shown to be effective atreducing mechanical hyperalgesia. However, intradermal administration ofKAI-1678 to a remote site was equally effective; suggesting that localadministration of KAI-1678 could provide system-wide pain relief. Thisconclusion is supported by the observation that KAI-1678 is effectivewith subcutaneous administration, either as a bolus or as a prolongedinfusion, to any site on the animal, including those distant from thesite of carrageenan injection. Although KAI-1678 appears to havesystem-wide activity, intravenous infusion of the compound, even at doserates sufficient to achieve plasma steady-state levels 5- to 10-foldgreater than those measured at the end of subcutaneous infusions thatachieve maximal efficacy, does not inhibit carrageenan-inducedmechanical hyperalgesia. Elucidation of the site-of-action for KAI-1678is ongoing, but two observations help to define the target of thecompound. Surgical disruption of the sciatic and saphenous nerves on theopposite hindlimb from the site of carrageenan injection abrogates theactivity of compound administered distal, but not proximal, to the siteof nerve disruption, suggesting a requirement for functionalinnervations of the injection site for the compound to achieve efficacy.Further studies indicate that an intact sympathetic nervous system and aadrenergic receptor signaling is required for KAI-1678 to be active whenadministered distant from the site of injury. One hypothesis arisingfrom these studies is that KAI-1678 acts on local nerves at the site ofinjection, perhaps within the dermis or epidermis, and, by anunidentified mechanism, elicits a descending pain-suppressing signalthat is dependent on a adrenergic receptor signaling.

Results similar to those obtained in the carrageenan-inducedinflammatory pain model were obtained in the L5 spinal nerve transectionmononeuropathic pain model used to investigate the efficacy of KAI-1678in neuropathic pain. In the L5 spinal nerve transection mononeuropathicpain model subcutaneous administration of KAI-1678 caused a reduction inthe level of injury-induced allodynia. However, although KAI-1678 wasactive in both neuropathic pain models, the maximum response and thedose required to achieve maximal response varied substantially in thetwo models. In these studies, the injury induced by L5 nerve transectionappeared to be much more susceptible to treatment with KAI-1678 in thatcomplete reversal of injury-induced allodynia was achieved with lowtotal doses of compound administered either as a subcutaneous bolus orinfusion.

Taken together, the nonclinical pharmacology studies support theactivity of KAI-1678 in inflammatory and neuropathic pain. These studiesalso suggest that the maximum effect and the dose required to achievemaximum effect may vary depending on the type and source of injurycausing the pain. Efforts are ongoing to identify the site of action andthe mechanism of action in each of these models.

Activity of KAI-1678 in the Carrageenan Inflammatory Pain Model

The rat carrageenan model has been extensively used to evaluate theresponse to modulators of inflammatory pain. In the model used for thestudies described below, a single 5 microL injection of a 1% solution ofcarrageenan into the plantar surface of the right hindpaw was used toelicit an inflammatory response resulting in local edema and mechanicalhyperalgesia. The mechanical hyperalgesia was quantitated as previouslydescribed using measurements of the nociceptive flexion reflex(Randall-Selitto paw-withdrawal test) in response to mechanical painfulstimulation at the site of carrageenan injection. Under the conditionsused in these studies, the paw withdrawal threshold (PVVT) in theRandall-Selitto test typically decreased from ˜90 g prior to carrageenaninjection to ˜55 g measured 1 hour after carrageenan injection for theuntreated animals. In the absence of further treatment, the PWT remainedstable at ˜55 g for several hours, making it possible to use this modelto evaluate the time-course of the response to treatment over the courseof at least six hours after the establishment of the disease state.

Intradermal Administration of KAI-1678

Previous publications have reported that local intradermaladministration of analogs of KAI-1678 is efficacious in thecarrageenan-induced inflammatory pain model. To determine whetherintradermal administration of KAI-1678 was active in this model, ratsinjected with carrageenan into the right hindpaw were treated one hourlater with an intradermal injection of 10 or 100 mcg/kg of KAI-1678either on the same (ipsilateral) hindpaw injected with the carrageenanor the other (contralateral) hindpaw. As shown in FIG. 18, intradermaladministration of 10 mcg/kg KAI-1678 to the ipsilateral site was able tocompletely reverse the mechanical hyperalgesia as indicated by the factthat the PWT returned to pre-carrageenan levels. Administration of 100mcg/kg KAI-1678 to the ipsilateral site was more effective than the 10mcg/kg dose, increasing the PWT beyond the pre-carrageenan levels andmaintaining the PWT at or above pre-carrageenan levels for a longerperiod. Strikingly, the effect of KAI-1678 demonstrated a rapid onset ofaction with the mechanical hyperalgesia substantially reversed fiveminutes after administration of the 10 mcg/kg dose and completelyreversed by the 5-min time point following administration of the 100mcg/kg dose.

Intradermal administration of KAI-1678 to the contralateral hindpaw wasalso able to completely reverse the mechanical hyperalgesia, with themaximum extent of reversal at each dose similar to that seen for thesame dose administered to the ipsilateral hindpaw (FIG. 18). Of note, asin the case in which KAI-1678 was administered close to the site ofcarrageenan injection, intradermal administration of KAI-1678 to thecontralateral paw resulted in a rapid onset of action with near orcomplete reversal of mechanical hyperalgesia at the 5-min time point forthe lower and higher doses, respectively. There was, however, adifference in the duration of the effect with the effect being moreprolonged at both dose levels when KAI-1678 was administered to theipsilateral hindpaw as compared to the contralateral hindpaw.

While the efficacy of local (ipsilateral) administration of selectivepeptidic εPKC inhibitors structurally related to KAI-1678 has beenreported previously, the demonstration that intradermal administrationof KAI-1678 to the contralateral hindpaw was similarly efficacious wasnot anticipated. This result suggests that local administration ofKAI-1678 at a remote site may elicit system-wide pain suppression basedin its ability to reduce mechanical hyperalgesia in an inflammatory painmodel.

Subcutaneous Administration of KAI-1678

KAI-1678 was administered subcutaneously to test whether administrationof the compound by this route could suppress mechanical hyperalgesia inthe carrageenan inflammatory pain model. As shown in FIG. 19,subcutaneous bolus administration of KAI-1678 on the thigh of thecontralateral hindlimb caused a dose-dependent suppression in the painresponse, with a dose as low as 0.1 mcg/kg completely reversingmechanical hyperalgesia for a short period of time. At doses up to 100mcg/kg the duration of the response increased. However, at doses greaterthan 100 mcg/kg the duration of the response decreased as the doseincreased, suggesting a parabolic dose-response for duration.

A group of animals was also dosed with 100 mcg/kg of KP-1723, theinactive control peptide (see above). As shown in FIG. 19, KP-1723 hadno effect on mechanical hyperalgesia, indicating that the effects seenwith KAI-1678 are not a consequence of non-specific effects of thecarrier moiety.

To determine whether the location at which KAI-1678 is administered as asubcutaneous bolus affects its activity in this model,carrageenan-injected rats were given a 10 mcg/kg subcutaneous bolus ofKAI-1678 either on the ipsilateral hindlimb close to the site ofcarrageenan injection, on the contralateral hindlimb, or on the centraltrunk above the contralateral hindlimb one hour after carrageenaninjection. Four hours later, each animal was given a second 10 mcg/kgsubcutaneous administration of compound to a different location as acontrol for possible animal-to-animal variability. The results shown inFIG. 20 indicate that the efficacy of the first dose of compound wassimilar regardless of the site of compound administration, and thatduration of the response with the second dose was reduced relative tothe first administration. This latter observation suggests that repeatedsubcutaneous bolus administrations may result in tachyphylaxis.Consistent with this observation, repeat administration of an □PKCinhibitor analog of KAI-1678 has shown a reduction in both the magnitudeand duration of the response that is inversely correlated with thelength of time between administrations (data not shown). The basis forthe apparent tachyphylaxis is under investigation.

A comparison of the results shown in FIG. 17 (intradermal dosing) withthose of FIG. 18 (subcutaneous dosing) indicates that both routes ofadministration cause a rapid response, with complete reversal ofmechanical carrageenan-induced hyperalgesia occurring as early as fiveminutes after dosing at the higher doses. However, the duration of theeffect following subcutaneous bolus administration was shorter than thatseen with intradermal administration adjacent to the carrageenaninjection site. In an attempt to extend the duration of theanti-hyperalgesic effect, KAI-1678 was administered as a subcutaneousinfusion to rats starting one hour after intradermal injection ofcarrageenan. As shown in FIG. 21, a subcutaneous infusion of KAI-1678extended the duration of the response in a dose-dependent fashion.However, the response to the subcutaneous infusion occurred in twophases. The first phase, which lasted approximately one hour, consistedof a rapid and complete reversal of the mechanical hyperalgesia at alldoses tested. The second phase, which developed typically between twoand three hours after the start of the infusion, was dose dependent withonly the higher doses (≧25 mcg/kg/hr) of KAI-1678 completely reversingmechanical hyperalgesia as indicated by a return of the paw withdrawalthreshold to pre-carrageenan baseline levels. At 250 mcg/kg/hr thereversal of pain was rapid and sustained without any recurrence ofhyperalgesia; although during the infusion a small decline in pawwithdrawal threshold was observed at ˜60 minutes after the start ofinfusion. Of note, a 25,000 mcg/kg/hr dose rate of KAI-1678 was testedin a subsequent subcutaneous infusion experiment. This dose rate, whichis 100-fold higher than the 250 mcg/kg/hr dose rate tested in theexperiment shown in FIG. 21, produced an effect comparable to that seenwith the 250 mcg/kg/hr infusion (data not shown). Therefore,subcutaneous infusion of KAI-1678 does not appear to display theparabolic dose-response observed with bolus subcutaneous administrationof the compound in this model (see FIG. 19).

At subcutaneous infusion rates ≧25 mcg/kg/hour the efficacy achievedduring the second phase was maintained for the duration of the infusionbut was rapidly reversed following the end of infusion (FIG. 21). Asdiscussed below, plasma levels of KAI-1678 declined at the end of asubcutaneous infusion but with a terminal half-life of ˜30 minutes. Thusthe decline in efficacy at the end of a subcutaneous infusion appears tobe more rapid than the drop in plasma concentrations, implying thatplasma concentrations are not the primary factor in determiningefficacy.

Intravenous Administration of KAI-1678

The intradermal and subcutaneous studies described above indicate thatKAI-1678 is efficacious when administered at a site remote from the siteof injury, suggesting that the compound may have a system-wide effect.We therefore investigated whether systemic administration of KAI-1678via the intravenous (IV) route was efficacious in the carrageenaninflammatory pain model. The IV infusion dose rates used in thesestudies were selected to achieve plasma levels of KAI-1678 that matchedor exceeded those associated with fully efficacious subcutaneousinfusions of KAI-1678 (e.g., ≧25 mcg/kg/hr) based on the results ofpharmacokinetic experiments (see below).

As shown in FIG. 22, IV infusions of KAI-1678 at rates as high as 1000mcg/kg/hr for as long as 5 hours had no effect on carrageenan-inducedmechanical hyperalgesia. Although plasma samples were not taken as partof these efficacy studies, pharmacokinetic studies suggest that plasmalevels of KAI-1678 at the 100 mcg/kg/hr IV dose rate would be comparableto or greater than those achieved with the mcg/kg/hr subcutaneousinfusion (FIG. 20), and that plasma levels of KAI-1678 at the 1,000mcg/kg/hr IV dose rate would be 5- to 10-fold higher than those achievedwith the 250 mcg/kg/hr subcutaneous infusion. The inability of IVKAI-1678 to affect carrageenan-induced hyperalgesia does not appear tobe due to any technical issues, since oral indomethacin, a knownanalgesic that has been shown to reduce carrageenan-inducedhyperalgesia, completely reversed the mechanical hyperalgesia whenadministered after the end of the 5-hr IV infusion of KAI-1678.Moreover, the lack of IV activity does not appear to be unique toKAI-1678, as structurally related εPKC inhibitors that were efficaciousin this model following intradermal or subcutaneous administration werenot active when administered as an IV infusion (data not shown).

Site of Action Studies with KAI-1678 in the Rat Carrageenan Model

The demonstration that KAI-1678 appears to elicit a system-wideresponse, but only when administered intradermally or subcutaneouslysuggested that the primary site of action for the compound might be inthe periphery.

This hypothesis, combined with the observation that the response toKAI-1678 administration was very rapid, even when the compound wasadministered to the contralateral limb, suggested that the afferentnerves in the skin at the site of compound administration might beessential for the rapid onset of action. Since signals propagated viathe peripheral afferent nerves in the skin on the contralateral hindlimbwould have to travel via the sciatic and saphenous nerves to affectmechanical hyperalgesia at the site of carrageenan injection on theipsilateral hindpaw, the need for intact sciatic and saphenous nerves onthe side of compound administration was investigated.

In the experiment shown in FIG. 23, a 1-cm section of the sciatic andsaphenous nerves was removed from the left hindleg of rats. The nextday, after the rats had recovered from surgery, carrageenan was injectedinto the plantar surface of the right hindpaw of the rats to elicit aninflammatory response. Sixty minutes after injection of carrageenan intothe right hindpaw, KAI-1678 was administered as a subcutaneous infusionto the left hindlimb at a site either proximal or distal to the sitewhere the 1-cm section of the sciatic and saphenous nerves had beenremoved. As shown in FIG. 23, a 25 mcg/kg/hr subcutaneous infusion ofKAI-1678 was effective at eliminating carrageenan-induced mechanicalhyperalgesia when the site of compound administration was proximal, butnot distal, to the site of nerve transection. Comparing with historicaldata (FIG. 21), the response to KAI-1678 administered proximal to siteof the transection of the sciatic and saphenous nerves was similar tothe response observed in surgically naïve rats (see FIG. 21), althoughthe onset of the second phase of activity appears to be more rapid inthe rats administered KAI-1678 as a subcutaneous infusion proximal tothe site of nerve transection. Although it is possible that othersecondary effects of the surgical procedure may have impacted theresponse to subcutaneous infusion distal to the site of nervetransection, the results of this experiment suggest that innervation atthe site of administration is required for KAI-1678 efficacy. As thesciatic and saphenous nerves contain motor neurons, primary sensoryafferent neurons, and sympathetic neurons, these data do not allow us todefinitively assign the site of action to one of these subsets ofneurons.

This finding suggests that 1678 may be acting on ‘normal’ neurons totransmit a signal centrally resulting in system-wide pain suppression.This modulation could be occurring in the dorsal horn; however, efficacyis observed when KAI-1678 is administered to sites outside of thedermatome affected by the pain stimulus, suggesting that system-widepain suppression is occurring above the level of the spinal cord (seebelow).

Earlier studies have reported that catecholamines from thesympathoadrenal system are involved in carrageenan-induced inflammatorypain and that εPKC is expressed in the sympathetic system. We thereforeinvestigated whether an intact sympathoadrenal system was required tomaintain the efficacy of distally administered KAI-1678. As shown inFIG. 24, bilateral lumbar sympathectomy plus suprarenal ganglionectomydid not alter the pain responses to carrageenan. However, the ability ofKAI-1678, administered as a 25 mcg/kg/hr subcutaneous infusion to thecontralateral hindlimb, to reverse the carrageenan-induced mechanicalhyperalgesia was abrogated in these animals. As shown in FIG. 24, boththe early and the later phases of the bi-phasic response to subcutaneousKAI-1678 infusion are eliminated by the sympathectomy and suprarenalganglionectomy. In previous experiments with □V1-2-containing analogs ofKAI-1678, we have also demonstrated that surgical sympathectomy, asdescribed above, eliminates the effect of εPKC inhibitors administeredas a bolus injection at a remote subcutaneous site, as well as at anintradermal site adjacent to the injury.

The sympathectomy experiments described above require surgicalintervention and recovery of animals for a week prior to interventionwith the carrageenan. As shown in FIG. 24, sham surgery, withoutsympathectomy, may have partially reduced the efficacy of KAI-1678.Consequently we sought to complement this study by investigating theeffect of pharmacological blockade of the sympathetic nervous systemfunction, using the alpha-adrenergic antagonist phentolamine, thuseliminating the need for surgery. Administration of this agent at thetime of carrageenan injection did not influence the establishment ofpain, although the effect of remote site subcutaneous bolusadministration of an □V1-2-containing analog of KAI-1678 was eliminated,as was the effect of an intradermal administration local to the site ofinjury (data not shown). Furthermore, the efficacy of an□V1-2-containing analog of KAI-1678 following subcutaneous bolusadministration in a second, neuropathic, pain model was also eliminatedby prior administration of phentolamine, although, again, thephentolamine did not prevent the establishment of pain in this model(data not shown).

Thus, surgical ablation of the sympathetic nervous system andpharmacologic blockade of alpha adrenergic receptors block theanti-hyperalgesic activity of εPKC inhibitors. It is not clear if theseeffects reflect a dependence on the sympathetic nervous system or onadrenergic receptor signaling in the CNS.

Summary of Carrageenan Inflammatory Pain Model Studies

The data above demonstrate that KAI-1678 can reverse inflammatory painwhen administered intradermally at the site of injury and intradermallyor subcutaneously at a remote site. Efficacy has not been observed withintravenous administration, even at KAI-1678 plasma levels equivalent tothose obtained during an efficacious subcutaneous infusion. We have alsodemonstrated that the remote site subcutaneous efficacy of KAI-1678 isdependent on innervation at the site of administration, which suggeststhat the remote-site efficacy of KAI-1678 is mediated by an effect onsubcutaneous neurons that are exposed to KAI-1678 at the site ofadministration. One hypothesis to explain these data is that, by actingon skin nociceptors, KAI-1678 elicits an ascending signal in the primaryafferent neurons that initiates a spinal or supraspinal reflex,ultimately resulting in descending pain suppression of thecarrageenan-induced hyperalgesia. Alternatively KAI-1678 might be actingby reducing tonic signaling in neurons at the injection site and thisinhibition might lead to the descending pain suppression. Descendingmodulation might involve alpha adrenergic signaling, which would beconsistent with the effect of phentolamine on KAI-1678 efficacy. Thus,in the spinal cord, nor epinephrine released from descending pathwayssuppresses pain by inhibitory action on alpha-2A-adrenoceptors oncentral terminals of primary afferent nociceptors (presynapticinhibition), by direct alpha-2-adrenergic action on pain-relay neurons(postsynaptic inhibition), and by alpha-1-adrenoceptor-mediatedactivation of inhibitory interneurons. This hypothesis contrasts withother models of εPKC action, in which a role has been proposed for εPKCin primary afferent nerve terminals modulating membrane channels andmembrane depolarization in response to inflammatory stimuli.

Activity of KAI-1678 in Neuropathic Pain Models

The activity of KAI-1678 has been evaluated in the L5 transection modelor modified Chung model, is a mononeuropathic pain model in which the L5spinal nerve is surgically transected, resulting in rapid development ofsustained pain. The activity of KAI-1678 in this model is describedbelow.

Activity of KAI-1678 in the L5 Spinal Nerve Transection MononeuropathicPain Model in Rats

The rat L5 spinal nerve transection model has been used to evaluate theresponse to modulators of neuropathic pain. In the studies describedbelow surgical transaction of the L5 spinal nerve was used to elicitmechanical allodynia, mechanical hyperalgesia and thermal hyperalgesiathat characterize neuropathic pain. In this model, the neuropathiceffects develop within a day and are reported to last for 1-2 months.

Mechanical allodynia and mechanical hyperalgesia are measured usingcalibrated von Frey filaments to elicit a flexion withdrawal response(paw withdrawal response) by pressing them against the plantar surfaceof the hindpaw on the same side as the nerve transection. The 2 g, 6 gand 10 g von Frey filaments are used to assess allodynia since thesestimuli are normally innocuous to naïve animals. The 15 g, 26 g and 60 gfilaments are used to assess algesia (response to noxious stimulus)since these stimuli elicit paw withdrawals in naïve animals and areconsidered to represent painful stimuli.

The existence of the disease state following nerve transection isconfirmed based on tests performed with the 2 g and the 10 g filaments.When tested with a total of 3 sets of 10 tests (30 tests total), thenumber of paw withdrawals in response to the 2 g von Frey filamenttypically increases from 1-2 prior to nerve injury to 12-15 pawwithdrawals following surgery. Similarly, the number of paw withdrawalsin response to the 10 g filament typically increases from 2-3 prior tonerve injury to 20-23 paw withdrawals following surgery. In the absenceof further treatment, the mechanical allodynia remains stable for atleast 3 weeks, making it possible to use this model to evaluate the timecourse of the response to treatment either as a bolus administration oran extended infusion, with sequential measurements typically over thecourse of 1-2 weeks after establishment of the disease state.

Once the disease state is confirmed, evaluation of the extent ofmechanical allodynia or hyperalgesia is determined by observing thenumber of paw withdrawals in responsse to 5 tests with each of the vonFrey filaments. Results are typically expressed either as the totalnumber of paw withdrawals for the allodynic filaments (2 g, 6 g and 10 gfor a total of 15 tests) or the algesic filaments (15 g, 26 g and 60 gfor a total of 15 tests), or as the lowest filament to which the animalsin a group have an average positive response defined as ≧3 withdrawalsout of 5 tests with a particular filament.

Along with measurements of the effects of compound treatment onmechanical allodynia and hyperalgesia, changes in the level of thermalalgesia in response to L5 spinal nerve transection have also beendetermined in this model using, the Hargreaves test. In these studies,rats are placed on a glass surface above a radiant heat source focusedon the lateral plantar surface of the affected hindpaw. When the heatsource is turned on, the glass surface heats up over time. The timeuntil the animal lifts its hindpaw (paw withdrawal latency, measured inseconds) is an indicator of thermal algesia. Under the conditions usedin this study, paw withdrawal latency decreases from 10-12 seconds priorto nerve injury to 6-8 seconds after L5 nerve transection, indicatingthe development of thermal hyperalgesia. In the absence of furthertreatment, the thermal hyperalgesia remains stable for at least threeweeks.

Subcutaneous Administration of KAI-1678

Rats given an L5 spinal nerve transection were treated with a bolussubcutaneous dose of KAI-1678 ranging from 0.00025-0.25 mcg/rat(˜0.001-1 mcg/kg) on day 7 post-transection. The degree of mechanicalallodynia and hyperalgesia was evaluated based on the number of pawwithdrawals (out of 30) in response to 6 different von Frey filamentsbefore and up to 3 hours after administration of the compound. Forcomparison, some rats were dosed with 0.250 mcg/rat (˜1 mcg/kg) KP-1723,the inactive analog of KAI-1678.

As shown in FIG. 25, KAI-1678 treatment resulted in a dose-dependentdecrease in mechanical allodynia and mechanical hyperalgesia at doses≧0.01 mcg/kg. Based on composite measurements with the 15 g, 26 g and 60g von Frey filaments (FIG. 25, left), KAI-1678 at 0.1 and 1 mcg/kgcompletely reverses mechanical hyperalgesia, as indicated by the returnto pre-surgery responses. Complete reversal of the hyperalgesia at thesetwo doses was observed between 30 and 60 minutes after compoundadministration, and an anti-hyperalgesic effect was observed for atleast 2 hours. In contrast to the results obtained with KAI-1678,KP-1723 treatment at 1 mcg/kg had no effect on mechanical hyperalgesia.Comparable results to those described above were obtained when the 2 g,6 g and 10 g von Frey filaments were used in order to measure KAI-1678effects on L5-transection-induced mechanical allodynia (data not shown).

The efficacy of KAI-1678 in this model was also apparent when pawwithdrawal threshold was measured by determining the minimum von Freyfilament to elicit 3 paw withdrawals out of 5 stimulations (FIG. 25,right).

To determine whether the efficacy of KAI-1678 in this model could beprolonged with continuous delivery, subcutaneous exposure, osmoticminipumps delivering 0.00025-0.25 mcg/day (˜0.001-1 mcg/kg/day) ofKAI-1678 or 0.25 mcg/day (˜1 mcg/kg/day) KAI-1723 were implantedsubcutaneously between the scapula on day 2 post-transection. Mechanicalallodynia was examined on Days 3, 5, 7, 9, 11 post-transection (Days 1,3, 5, 7, 9 post-pump implantation) using the 2 g and the 10 g filaments.As shown in FIG. 26 (left), results from the tests with the 10 gfilament indicate that KAI-1678 dose rates ≧0.0025 mcg/day (˜0.01mcg/kg/day) are able to substantially reduce the level of mechanicalallodynia, with the 0.025 and 0.25 mcg/day (˜0.1 and 1 mcg/kg/hr) doserates producing nearly complete reversal of mechanical allodynia thatlasted until Day 7 post-pump implantation. At the highest dose rate,evidence of reduction in mechanical allodynia was observed as late asDay 11 post-transection. Similar results were obtained using the 2 gfilament to evaluate mechanical allodynia (data not shown) and using theHargreaves test to thermal hyperalgesia (FIG. 26, right). After about 5days of complete reversal of hyperalgesia in the presence of pumpscontaining KAI-1678, we noted a loss of efficacy. This was not due to atolerance to the drug, as a subcutaneous bolus administration of thedrug at this time elicited complete reversal of pain (data not shown)and it may be that the loss of effect was due to a failure of the pumpsto maintain delivery.

Summary of Neuropathic Pain Model Studies

The data above demonstrate that KAI-1678 is extremely effective in theL5 spinal nerve transection mononeuropathic pain model. The potency ofKAI-1678 administered as a subcutaneous bolus is comparable in reversingmechanical and thermal allodynia in the L5 spinal transaction model.When administered by subcutaneous infusion, KAI-1678 is about 1000-foldmore potent than in the carrageenan-induced inflammatory pain model. Thereason for the high sensitivity of the L5-transection model to KAI-1678treatment is not known, but it likely reflects the responsiveness ofthis model to selective εPKC inhibitors, since this model is exhibits asimilar high sensitivity to structurally-related analogs of KAI-1678.The demonstration that KAI-1678 has activity in two neuropathic painmodels suggests that this compound may have clinical utility for themanagement of neuropathic pain in humans.

Conclusion from Rat Pharmacology Studies

The activity of KAI-1678 has been evaluated in rat inflammatory andneuropathic pain models. Subcutaneous administration of the compound isefficacious in the models shown. The potency of KAI-1678 administered bysubcutaneous bolus is similar across the models, as is the rapid onsetand duration of effect tested, although the overall effect size doesvary between models. The response to different infusion dose rates isalso different between models. In particular, the L5 transectionmononeuropathic pain model appears to be particularly sensitive toKAI-1678 with doses approximately one thousand fold lower being activein this model than in the carrageenan model.

While experiments with these models are continuing, additional work isbeing done in other pain models. In particular, preliminary results inthe Brennan incision model and the single spared nerve model suggestthat KAI-1678 is also active in these models at doses similar to thosethat are active in the carrageenan model.

Taken together, the available animal efficacy data suggest that KAI-1678may be active in a variety of pain models and support the continueddevelopment of the compound.

Pharmacokinetics in Animals

The pharmacokinetics of KAI-1678 are being studied in rats administeredthe compound as an intravenous bolus and infusion or as a subcutaneousbolus and infusion. The toxicokinetics of KAI-1678 are being studied indogs administered the compound as a subcutaneous infusion as part of thetoxicity studies. In these studies, plasma levels of KAI-1678 have beendetermined using a sandwich-based ELISA assay developed at KAIPharmaceuticals. The lower limit of quantitation of this assay isapproximately 0.3 ng/mL. A similar assay has been developed to measuretissue levels of KAI-1678.

In rats KAI-1678 is rapidly cleared from systemic circulation followingintravenous bolus administration. When administered as a 2-hrintravenous infusion at 50 mcg/kg/hr, steady state plasma concentrationsof approximately 10-20 ng/mL appeared to be achieved by 60 minutes, anddropped off rapidly after the end of infusion. The terminal half-livesfor the bolus and infusion administrations of KAI-1678 via theintravenous route are approximately 50 and 25 minutes, respectively.

Subcutaneous administration of KAI-1678 to rats as either a bolus(approximately 80 or 800 mcg/kg) or as an infusion (19 and 190mcg/kg/hr) resulted in dose-proportional increases in exposures over thedoses tested. Plasma concentrations of KAI-1678 reached maxima atapproximately 10 minutes and 1 hour for the subcutaneous bolus andinfusions, respectively, and then decreased with a half-life ofapproximately 35 and 45 minutes, respectively, once the peptide was nolonger being administered. The bioavailability of KAI-1678 administeredby the subcutaneous route is approximately 10% in rats.

Although the terminal half-life was not determined, KAI-1678 was alsorapidly cleared from the systemic circulation in dogs administeredKAI-1678 following an extended subcutaneous infusion over 5 days. Inthis study, KAI-1678 was detected in tissue samples from a variety oforgans removed at the end of infusion (most notably the kidney, liverand lung), while levels of KAI-1678 in brain and spinal chord wereconsistently low.

Rat Pharmacokinetics

Intravenous Administration

To initially characterize the pharmacokinetics of KAI-1678 in rats, malerats were administered KAI-1678 at doses of 100 and 1,000 mcg/rat (˜300and ˜3,000 mcg/kg) as an IV bolus injection via the tail vein. Bloodsamples were withdrawn periodically for up to two hours following theinjection for plasma analysis of KAI-1678 levels. There were three andtwo animals in the 100 and 1,000 mcg dose groups, respectively, andthere was consistency in KAI-1678 plasma concentrations between animalsin each dose group. As shown in FIG. 27, C_(max) plasma levels ofKAI-1678 increased in a dose-proportional manner over the doses tested.Preliminary analysis of these data indicates a terminal half-life ofapproximately 38 and 66 minutes for the ˜300 mcg/kg and the ˜3,000mcg/kg dose levels, respectively.

Subcutaneous Administration

The majority of the animal efficacy studies have been performed withKAI-1678 administered to rats as a subcutaneous bolus or infusion.Therefore, the pharmacokinetics of KAI-1678 administered by thesubcutaneous route has been evaluated based on measurements of plasmaconcentrations of KAI-1678.

In the experiment shown in FIG. 29, male rats were administered KAI-1678as a subcutaneous bolus injection (200 microL into the left hindleg) atdoses of 25 or 250 mcg/animal (approximately 80 and 800 mcg/kg,respectively). Blood samples were taken at various time points over twohours after administration of the compound to determine plasmaconcentrations of KAI-1678. There were three and four animals in the 25and 250 mcg/animal dose groups, respectively, and the KAI-1678 plasmaconcentration measurements were consistent between animals in each dosegroup. C_(max) occurred within 10 minutes after injection and increasedin a dose-proportional manner over the two doses tested. A preliminaryestimation of terminal half-life from these data is 35 minutes for bothdose levels. A comparison of the areas under the plasma concentrationversus time curves for KAI-1678 administered as an intravenous bolus andas a subcutaneous bolus indicates that the bioavailability of KAI-1678administered via the subcutaneous route is approximately 10% in rats atthis site of administration.

In the experiment shown in FIG. 30, male rats were administered KAI-1678as a continuous subcutaneous infusion for 2 hours at doses of 19 and 190mcg/kg/hr. Blood samples were taken at various time points throughoutthe infusion and for 1 hour after the end of infusion to determineplasma concentrations of KAI-1678. There were two animals per dose groupand the plasma KAI-1678 concentration measurements were consistentbetween animals in each dose group. At the lower dose rate (19mcg/kg/hr), plasma concentrations reached steady state (˜3 ng/mL) by 60minutes and remained relatively constant throughout the rest of theinfusion. At the higher dose rate (190 mcg/kg/hr), plasma concentrationsof KAI-1678 rose throughout the infusion and did not appear to reach aplateau, or steady state, within the two hours of infusion (C_(max)˜60ng/mL). At both dose rates, plasma concentrations of KAI-1678 droppedquickly after the end of infusion although compound was still detectableat both dose levels one hour after the end of infusion.

A comparison of the data in FIG. 28 and FIG. 30 indicates that a 50mcg/kg/hr intravenous infusion of KAI-1678 will produce plasmaconcentrations of compound (10-20 ng/mL) that are higher than thoseachieved with a subcutaneous infusion at 19 mcg/kg/hr (˜3 ng/mL) andnearly at the level achieved with a subcutaneous infusion at 190mcg/kg/hr (˜60 ng/mL). Interestingly, there does not seem to be acorrelation with plasma concentration and efficacy, as efficacy is notobserved with iv administration of KAI-1678 even at dose rates higherthan those required to achieve maximal efficacy via subcutaneousinfusion of the compound.

Dog Pharmacokinetics

As part of a range-finding toxicology study, 6 beagle dogs (one per sexper group) were administered continuous subcutaneous infusions ofKAI-1678 for 5 days at dose levels of 3, 8 and 25 mg/kg/day. Bloodsamples were collected at various times on Day 1 (start of infusion) andDay 6 (after the end of infusion). Plasma concentrations of KAI-1678increased over the course of the first four hours of infusion, and inmost cases increased another two- to three-fold by the end of infusionon the fifth day, suggesting that steady state was not reached within 4hours of subcutaneous infusion at the dose levels tested (FIG. 31).However, at all dose levels, plasma levels declined quickly uponcessation of infusion although terminal half-life was not calculatedbecause of the small number of data points available.

As part of this study, tissues were harvested from a subset of theanimals at the end of dosing. Tissue extracts were prepared and analyzedfor the presence of KAI-1678, which was detected in a subset of tissuesfrom the two highest dose groups: liver, lung, kidney, brain (cerebrum),spinal cord, injection site (consisting of skin and underlying skeletalmuscle), a peripheral nerve in the left fore-limb, and muscle in the leg(not proximal to infusion site). In general, tissue levels of KAI-1678in the major organs (kidney, liver and lung) increased with increasingdose and reflected differences that were seen in plasma levels. Asexpected, KAI-1678 concentrations were highest in the infusion site skin(i.e., for 25,000 mcg/kg/day animals), although the underlying musclehad relatively low levels. KAI-1678 levels in nervous system (spinalcord and ulnar nerve) and peripheral tissues (muscle and skin) were lessconsistent between animals and dose groups. The levels in the brain andspinal cord were, for the most part, consistently low, in many casesjust above the limit of quantitation, indicating relatively low exposureof these tissues to subcutaneously infused KAI-1678.

TABLE 12 Tissue Levels of KAI-1678 in Dogs Administered KAI-1678 as aSubcutaneous Infusion at 25 mg/kg/day for Five Days Animal 4001 Animal4501 (25 mg/kg/day (25 mg/kg/day male) female) Mean (ng KAI- Std. Mean(ng KAI- Std. 1678/g tissue) Dev. 1678/g tissue) Dev. Brain   6* 1.2 71.3 Spinal Cord  11 1.3 140 13 Peripheral Nerve 429 56 155 14 Lung 25422 1364 150 Liver 224 15 1505 176 Kidney 689 84 3054 522 Skeletal Muscle133 20 33 4 Dose Site Muscle 111 14 1003 155 Dose Site Skin 1139  886165 998 *includes at least one data point that was below the specifiedlimits of quantitation.

The invention claimed is:
 1. A hybrid peptide construct, comprising: afirst peptide comprising the amino acid sequence of SEQ ID NO:58; asecond peptide comprising the amino acid sequence of SEQ ID NO:1; and acarrier peptide, wherein the first peptide is covalently linked to thesecond peptide.
 2. The composition of claim 1, wherein the carrierpeptide comprises the amino acid sequence of SEQ ID NO:59.
 3. Thecomposition of claim 1, wherein the intracellular carrier peptidefurther comprises the amino acid sequence of SEQ ID NO:4.
 4. Thecomposition of claim 2 or 3, wherein the carrier peptide is modified atits N-terminal end by an acyl, alkyl, or sulfonyl group.
 5. Thecomposition of claim 4, wherein the modification is an acyl group. 6.The composition of claim 2 or 3, wherein the carrier peptide is modifiedat its C-terminal end.
 7. The composition of claim 6, wherein themodification is formation of an amide.
 8. The composition of claim 1,wherein the first peptide further comprises a terminal Cys.
 9. Thecomposition of claim 8, wherein the terminal Cys is located at theC-terminus of the first peptide.
 10. The composition of claim 8, whereinthe terminal Cys is located at the N-terminus of the first peptide. 11.The composition of claim 8, wherein the carrier peptide furthercomprises a terminal Cys.
 12. The composition of claim 11, wherein thecarrier peptide is linked to the first peptide via a disulfide bond. 13.The composition of claim 1, wherein the second peptide further comprisesa terminal Cys.
 14. The composition of claim 13, wherein the terminalCys is located at the N-terminus of the second peptide.
 15. Thecomposition of claim 13, wherein the terminal Cys is located at theC-terminus of the second peptide.
 16. The composition of claim 13,wherein the carrier peptide further comprises a terminal Cys.
 17. Thecomposition of claim 16, wherein the carrier peptide is linked to thesecond peptide via a disulfide bond.